•
Mechanism of Selective Incorporation and Genomic Placement of
Primer tRNALys3 into Buman Immunodeficiency virus Type 1
by
Johnson Mak
A the sis submitted to ths Faculty of Graduate Studies and Research,
McGill Univsrsity, in partial fulfillment of the requirements of the
degree of Doctor of Philosophy
Department of Medicine, Division of Experimental Medicine
McGill University, Montréal, Canada.
March 1996
•
~
Johnson Mak, 1996
i
1+1
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•
Ta rny parents, for their infinite support,
understanding, and encouragement.
Tc my sister, for her humors, and for jusl
being there.
To my brother.
has
for his achievement, which
encouraged me
ta strive
for my own
success.
But above aIl,
this thesis is dedicated ta
aIl those who lost their lives or a
one in this tragedy called AlOS .
•
ii
loved
•
Abstract
During
HIV-l
assembly.
the
major
marnmalian
isoacceptors,
tRNALys
tRNA Lyol.2 and tRNAL y n3, are selectively incorporated into the virus.
project has been to study the mechanism involved in this process.
My
This
was done by transfecting COS-7 cells with wild type and mutant HIV-1
proviral
process.
is
DNA,
the
effects
of
these mutations
on
this
In the wild type virus, 60% of viral low molecular weight RNA
tRNALy..
packdging
and analyzing
compared
to
6%
tRNALys
is
selective.
of
moleculcs of tRNAL y s3,
Mutations
which
including
the
of
the
tRNA
in
HIV-l
the cytop1asm.
cantains
i. e,
the
approximately 8
the primer for reverse transcriptase, per virion.
either
primer
remove
binding
the
S'
site,
portion
or
which
of
the
reduce
RNA
genome,
l~~A
genornic
incorporation approximate1y la fo1d, have no effect upon select tRNALys
incorporation,
indicating that genonic RNA does not play a role in this
prr)cess.
the
On
Pr160Qag-pol
reduces
molecule/virion,
evidence
carrying
other
i.e,
implicating
hand,
tRNALys)
inhibits
a
large
deleticn
of
RT
sequences
incorporation
to
approximately
select packaging
of
tRNALys.
in
1
Initial
Pr160gag-pol as being the viral precursor protein
tRNALYs
into the virus is as fol1ows:
1) Viral partic1es
composed on1y of PrSS gog do not se1ective1y package tRNALys, whi1e viral
partic1es composed of both prSS gog and Pr160gog-pol do.
2) Immature
viral particles,
unable to process these precursor proteins due to an
inactive viral protease.
that
processing
of
still show select tRNALys packaging,
precursor
proteins
is
not
indicating
required.
We
next:
investigated regions within Pr160gog-pol which may play a ro1e in the
tRNALy. packaging process
using mutationa1
ana1ysis.
The
carboxy1
deletion of IN sequences in Pr160gog-po1 does not significantly affect
select tRNALys packaging, but carboxy1 de1etions which include both the
IN sequence and the RNase H and connection domains of RT do.
Smal1
ami no acid insertions(2-3) placed into various domains of RT show no
effect upon tRNALys packaging when p1aced inco the fingers. palm, part
of the thumb.
and RNase H domains
• but mutations within or just N-
terminal to the connection domain inhibit select tRNALys packaging.
•
A
direct correlation has been found between these mutations which affect
tRNALys) packaging and the absence of mature Gag and Gag-Pol pro teins in
iii
•
the
virus.
tRNALYs
Since
precursor
packaglng,
it
seems
incorporation into the virus
packl;Jing
into
the
virus
as
processing
likely
is
that
the
is due to the
a
result
not
of
required
inhibition
inhibi tion of
these
for
select
of
tRNAL~'l1
Pr160gaa-pol
connection
domdin
mutations. We have aiso done preliminary experiments on the effect of
sorne of these mutations upon tRNALys3 :>lacement on the viral RNA genome.
Genomic placement of tRNALysJ in vivo was detected by measuring reverse
transcription in vitro,
source
of
using exogenous RT and total viral RNA as the
primer/ternplate.
Our
results
indicate
placement of tRNA Lys3 occurs independently of viral
that
the
genomic
precursor protein
processing and the presence of IN sequences. Genomic placement was found
to be significantly reduced when one of the connection domain mutations,
SVC21 GR, was tested .
•
iv
•
Résumé
Lors de l'assemblage du virus VIH de type I,
de
mammi fère
l' ARNtLysl, 2 et l' ARN t Ly s3
l' ARN t Lys.
façon sélective par le virus.
mécanismes
les isoaccepteurs majeurs
sont
incorporés
de
Le but de mon projet était d'étudier les
impliqués dans ce processus d'incorporation en transfectant
des cellules COS-7 avec de l'ADN proviral de type sauvage ou muté et en
analysant
les
effets
cl incorporation.
Chez
1
l'ARNtLYS
de
le
sélective
est
de
de
mutations
type
celui-ci
sur
sauvage,
compose
la
de
60%
ce
processus
présence
de
l' ARN viral de
tandis que dans le cytoplasme on en retrouve
6%.
Chaque particule virale contient environ 8
l' ARN t Ly s3. l'amorce nécessaire pour la transcriptase
propart ion
molécules
virus
car
faible poids moléculaire.
une
différentes
de
inverse (TI).
Des mutations qui enlèvent le bout 5' du génome viral et
en
le
incluant
l'incorporation
site
de
de
liaison
l'ARN d'un
de
l'amorce
facteur
de
10
ou
n'ont
qui
pas
réduisent
d'effet
sur
l'incorporation sélective de l'ARNtLYS, ce qui semble indiquer que llAkN
génomique n'affecte pas ce processus.
Cependant une délétion dans la
séquence de la TI du préc\\rseur Pr160 Qa g-pol r(dui t
a
l'ARNtLYs
environ
impliquaient
transportant
déjà
le
molécule
l'ARNtLYS
l'ARNtLYS alors que
Pr160 0AO -pol le font.
le
par virion.
pr160gag-pol
dans
uniqueme~1t du
contenant
maturer
une
le
PrSS gag
comme
virion:
l'incorporation du
Plusieurs
étant
le
évidences
précurseur
1)
Les
particulp.s
n'incorpore
pas
de
viral
virales
façon
sélective
les particules virales contenant le PrSS gag et le
2) Les particules virales qui sont incapables de
précurseur parce qu'ils possédent une protéase
inactive,
démontrent une incorporation sélective de l'ARNtLys indiquant que cette
maturation nlest pas nécessaire.
au
niveau
des
régions
Nous avons donc investigué davantage
Pr160gag-pol
Douvant
jouer
un
l'incorporation de l'ARNtLyS en utilisant des mutations.
du
bout
C-terminal
de
la
séquence
intégrase(INI
du
rôle
dans
Des délétions
Pr1600AO-pol
n' af fectent pas l'incorporation de l' ARNtLys tandis que des délétions
éliminant la séquence IN, la RNase H et le domaine de la liaison avec la
•
TI affectent ce processus.
De petites insertions (2-3) d'acides aminés
lorsque placées dans les différents domaines de la TI comme la paume,
les doigts, une partie du pouce et le domaine RNase H produisent aucun
v
•
effet
alo~s
que des insertions situees â
terminale du domaine de
liaison inhibe
l'int~ri~ur
ou du
cot~amino
l'incorporation selective des
Une corrélation direct.e fut démontrée entre les mutations qui
ARNtLys.
affectent l'incorporation de l'ARNtLYs dans le virus et la maturation du
précurseur Pr160 0a q-pol dans le virus.
Pr160gb.g-pol n'est pas nécéssaire à
virus,
il
Comme la maturation du précurseur
l'incorporation des ARNtLYs dans
semble donc que cette inhibition est causée par
It:'
l'absence
d'incorporation du précurseur Pr160gag-pol dans le virus de à la présence
des mutations dans le domaine de liaison.
Nous avons aussi conduit des
études sur l'effet de ces mutations pour la mise en place de l'ARN t LysJ
sur le génome dlARN viral.
transcriptase
In vivo nous avons mesuré l'activité de la
inverse alors qu'in vitro nous avons utilisé de
la 'rI
exogène et de l'ARN viral comme source cl 1 amorce/patron.
Nos résultats
indiquent que la mise en place de l'ARN t Lys3 se produit indépendamment
de
la maturation du précurseur ou de
Cependant celle-ci
est
fortement
la présence des séquences
réduite
domaine de liaison. soit la SVC21 GR .
•
vi
IN.
avec une des mutations du
•
Acknowledgements
l would like ta
expres~
my appreciation tO aIl the past and the present
staff members in the Lady Davis Institute.
to
the
prin~ipal
5nvestigators.
A
from the house keeping staff
simple
thanks
or
a
list
individuals' names will not be sufficient to express rny gratitude.
of
The
last five years at the LDI has not only been a time for science, but it
was aiso a personal enrichrnent process.
There were times that l wish l
was r.ot there to wi.tness or to participate in, but there were aise times
that 1 was glad to partake in.
For aIl those people with whom 1 have
come across over the past five yeal's,
l
thank you.
In my heart,
are bits and pleces from each and everyone of you which l
with me no matter where l go.
there
will bring
In a way you have aIl shaped me to be the
persan 1 am today(No ius;.\l.t intenùed).
1 will net provide a list of individuals te whom l ewe my gratitude, but
you know who you are.
For those who have helped me along the way, a
brief mention can never repay for aIl their assistance.
However,
individuals have to be singled out
They are my
supervisor,
Dr.
Lawrence Kleiman,
from this process.
and my colleague,
as
weIl
two
as my
friend, Dr. Mark A. Wainberg.
Larry,
how
~hould
l
begin?
In the past five years,
tremendously great superviser as weIl
you have been a
a Hreat friend.
dS
wou Id have
1
hoped that certain things could have been different, but above aIl 1 am
glad that 1 had the privilege to obtain my Ph.D.
supervision.
l
training under your
would be honored te study under your supervision if l
had to do it aIl over again.
1 thank you for your faith,
trust, and those not so great pep-talks.
confidence,
1 especially tbank you for
giving me the freedom to take on my research direction and to establish
coll~boration
with members of the institute in my own free will.
importantly, you have talked me the essence of intelligence.
shawn me that
You have
the essence of intelligence is not ba.sed on how much
knowledge one has,
•
Most
but rather,
how willingly one has the courage to
openly accept how little we aIl know,
criticism with open arrns.
and be humble enough to greet
For that, 1 thank you!
vii
•
Mark,
l
thank you!
Without
them.
possible.
a
Thank you for yaur Eaith,
lot
of
But especially,
work
l
in
this
thesis
confidence,
would
and support.
never
have
been
thank you f_r yaur helping hands, without
which 1 would have never been able to complete my Ph.D. training in this
institute.
l aIse wish to acknowledge Health and Welfare Canada for having provided
me with a Pre-Doctoral Fellowship for rny graduate training.
A special thanks goes to Normand Pepin for translating the abstracto
Last,
but certainly not least.
Thanks to Sunita de Tourreil for her
support and friendship during the writing of this Thesis .
•
viii
•
preface
In
with
dccc)l"danCB
the
Guidelines
for
Thesis
Preparation
fram
Department of Graduate Studies and Research at McGill University.
candidate
has
exercised
n~n~script-based
for
option
of
writing
the
thesis
the option of including,
as
part of
the
the text of a paper(s) submitted or to be submitted
publication,
published
or
paper (s).
the
clearly-duplicated
These
texts
must
be
text
bound
of
as
a
an
integral part of the thesis.
1f
this
option is
lo~ical brid~es
chosen,
connecting texte
that
provide
between the different papers are mandatory.
1'he thesis must be written in such a way that it is more
than
a
mere
collection
of manuscr ipts;
in
ether words,
results of a series of paper must be integrated.
'rhe thesis must still conform to aIl ether requirements of
the "Guidelines for Thesis Preparation".
include:
A Table of Contents,
The thes!s must
an abstract in English and
French, an introduction which clearly states the rationale
and objectives of the study, a comprehensive review of the
literature,
a
final conclusion and summary,
and a
through
bibliography or reference list.
Additional material must be provided where appropriate(e.g.
in appendices) and in sufficient detail to allow a clear and
precise
judgment
to
be
made
of
the
importance
and
originality of the research reported in the thesis.
In the case of rnanuscripts co-authored by the candidate and
•
others,
the
candidate
is
required
to make
an
explicit
atatement in the theaia as to who contributed to auch work
and to what extent.
Supervisors must attest to the accuracy
ix
The
as
thesis.
Candidates have
thesis,
the
the
a
•
of such SLdtement dt the doctoral
or~l
d~t~nse.
task of the exarniners is made more ditticult
it 1S in
the candidate's
interest
Und.er
no
circumstanc8S
CdS~S,
these
ln
tht""'
to make per::ectly clear
the responsibilities of aIL the authors of
papers.
Si.nC'~
cau
a
th~
c0-author~d
co-author ot
Any
component of Buch a th8sia serve .s an examiner t'or that
thesis.
l
have included,
as chapters of
paper which has been published.
this thesis.
the texts of an originel1
one original rnanuscript which
hdS
b~en
subrnitted for publication, and sorne preliminary data which are included
in the format of a short communication.
own Abstract,
and
Chapters 2 and 3 include their
Introduction, Materials and Methods,
Reference
sections.
Chapter
4 has
its
Results,
Discussion.
own abstract,
and
the
Introduction, Materials and Methods, Results, and Discussion are written
in a single section.
the
thesis
have
respectively.
A General Introduction and General Discussion to
been
inl..:luded
and
represent
Chapters
In order to bridge connecting papers,
1
'lnd
5,
Chapters 2 to 4
each contain a preface.
The manuscripts presented in the thesis are the following:
Chapter 2.
Mak, J., Jiang, M., Hammarskjold, M.L., Rekosh, D"
Kleiman,
L.
1994.
Incorporation
Particles.
of
Role of
tRNALys
Pr160gag-pol
into
Journal of Virology.
Human
in
Wainberg, M.A"
Mediating
the
and
Selective
Immunodef iciency Virus
Type
1
68:4. 2065-2072.
Chapter 3.
Mak, J., Cao, Q., Huang, Y., Lowy, I., Prasad, V.R .. Wainberg, M.A., and
Kleiman, L.
1996.
Reverse Transcriptase Connection Domain Mutations in
Prl60gag-pol Inhibit the Select Incorporation of Primer tRNALy.J
•
HIV-I.
Journal of Virology.
Submitted for Publication .
x
into
•
Chapter 4 .
Huang, Y., Li, Z., Wainberg, M.A., and Kleiman, L.
Mak, J.,
Genomic
Placement of HIV-l
Func~i~nal
1996.
The
Primer tRNALyo3 Occurs in the Absence of a
Protease or Integrase Sequences.
The candidate was responsible for all the research described in Chapters
2 to 4.
Dr.
M.Jiang provided initial assistance in the 2-D PAGE
analysis in chapter 2,
Dr.
Q.Cao provided technical assistance in sorne
of the plasmids isolation and virus isolation in chapter 3, Dr. Y.Huang
provided
genomic
technical advice on the western analysis and
placement
in
Assay
chapter
3
&
4
the
respectively.
in
vi tro
Dr.
Z.Li
provided the enzyme reverse transcriptase used in the in vitro genomic
placement assay in chapter 4.
Ors.
M.-L.Hammarskjëld,
O.Rekosh,
V.R.Prasad, I.Lowy provided sorne of the mutants used in chapters 3 & 4.
Dr. M.A.Wainberg provided the containment facilities, many reagents, and
many helpful advice throughout this study.
AIl this work was done under
the supervision of Dr. L.Kleiman.
Other relevant studies that the candidate was involved with, but are not
included as part of the thesis.
1. Jiang, M., Mak,
J.,
Wainberg,
M.A.,
Parniak,
M.,
Cohen,
E"
and
Kleiman, L. 1992. A Variable tRNA Content in HIV-1 IIIB. Biochemical and
Biophysical Research Communication. 185:3, 1005-1015.
2. Jiang, M., Mak, J., Ladha, A., Cohen, E., Klein, M., Rovinski, B"
and Kleiman,
Type
and
L. 1993. Identification of tRNAs Incorporated into Wild
Mutant Human
Immunodeficiency Virus Type
1.
Journal
of
Virology. 67:6, 3246-3253.
3. Arts, E., Mak.
J.,
Kleiman,
L.,
and Wainberg, M.A.
1993. Mature
Reverse Transcriptase (p66/p51) is Responsible for Low Levels of Viral
•
ONA Found in Human Immunodeficiency Virus Type l(HIV-l). Leukemia. 8:1,
175-178 .
xi
•
4.
Jiang,
M ..
Mak,
J ••
Wainberg,
M.A.
and Kleiman,
L.
1993.
Reverse
Transcriptase is an Important Factor for the Primer tRNA Selection in
HIV-1. Leukemia. 8:1, 149-151.
5. Arts, E., Mak, J., Kleiman, L., and Wainberg, M.A. 1994.
DNA Found
in Human Immunodeficiency Virus Type 1 Particles 1s Not Required for
Viral Infectivity. Journal of General Virology. 75, 1605-1613.
6.
Li,
X.,
Parniak,
Maki
M.A.
J.,
1994.
Arts,
Effects
E.J.,
Kleiman,
L.,
of
Alterations
of
Wainberg,
Primer
Sequences on HIV-1 Replication. Journal of Virology.
M.A.,
and
Binding Site
68:10, 6198-6206.
7. Huang, Y., Mak, J., Cao, Q., Li, Z., Wainberg, M.A., and Kleiman, L.
1994. Incorporation of Excess Wild Type and Mutant tRNALys3 Into HIV-1.
Journal of Virology.
68:12, 7676-7683.
8. Huang, Y., Shalom, A., Li, Z., Wang, J., Mak, J., Wainberg, M.A., and
Kleiman,
L.
1996.
Effects
of Modifying
the
Initation of HIV-1 Reverse Transcription.
tRNALys3
Anticodon upon
1996. Journal of Virology.
Submitted for Publication.
9. Mak, J., Wainberg, M.A., and Kleiman, L. 1996. The Incorporation of
Cyclophilin A into Human Immunodeficiency Virus Type 1 Is Independent
from Primer tRNA and Genomic RNA Packaging. Biochemical and Biophysical
Research Communication. Submitted for Publication.
10. Li, Z., Arts, E., Huang, Y., Mak, J.
Kleiman,
L.
1996.
Multiple Forms of
Publication .
•
xii
f
Cao, Q., Wainberg, M.A., and
tRNALys3
in HIV-l.
Sumitted for
•
Table of Contents
Page
..
iii
R6sum6 ••••••••.•••••••••••••••.•••••••••••••••••••••••••
v
Acknowledgemsnts ••••••••••••••••••••.•••••••••••••••••••
vii
Preface to the Thesis ••••••••••••••••••••.••••••••••••••
ix
Abstract
..
xiii
List of Figures and Tables ••••••••••••••••••••••••••••••
xvi
List of Abbreviations •••••••••••••••••••••••••••••••••••
xviii
Table of Contents
Chapter 1: Literature Review ••••••••••••••••••••••.••••
1
1.1. Introduction ••••••••••••••••••••••••••••
2
1.2. Retrovirus Classification •••••••••••••••
3
1.3.1. Human Immunodeficiency virus Structure
and Genome organization•••••••••••••••••
4
1.3.2. OVerview of HIV-l Replication Cycle ••••
7
1.4. HIV-l Viral Assembly: Viral Proteins, Viral
RNA, and Their Roles in Viral Assembly ••
13
1.4.1. Viral Proteins •••••••••••••••••••••
14
1.4.1.1. PrSS craCl
..
14
1.4.1.1.1 Matrix ••••••••••••••••••••••
16
1.4.1.1.2. Capsid ••••••••••••••.••••••
17
1.4.1.1.3. p2 •••••••••••••••••••••••••
18
1.4.1.1.4. Nucleoeapsid •••••••••••••••
18
1.4.1.1.5. p6
21
..
1.4.1.2. Pr160 gag-pol
•
..
22
1.4.1.2.1. Expr.ssion •••••••••••••••••
22
1.4.1.2.2. Enzymes coded by Pr160gall'-pol
24
1.4.1.2.2.1. Prot••so ••••••••••••••••
24
1.4.1.2.2.2. Reverse Transcriptase •••
25
1.4.1.2.2.3. Int.gr.sa
.
26
1.4.1.3. Env.lape •••••••••••••••••••••••
28
1.4.1.4. Naf
..
29
1.4.1.5. vif
..
30
xiii
•
1.4.1.6. vPr ••••••••••••••••••••••••••••
1.4.2. Virion Associated RNA ••••••••••••••
1.4.2.1. Ganomic RNA ••••••••••••••••••••
1.4.2.2. tRNALy.3 ••••••••••••••••••••••••
1.4.2.2.1. Retrovira1 Primer tRNAs ••••
32
33
33
35
35
1.4.2.2.2. rnteraction of Primer
tRNALy.3 and Ganomic RNA ••••••••••••
38
1.4.2.2.3. rnteraction of Primer tRNALY·3
with Viral Proteins •••••••••••••••
40
1.4.2.2.4. Ro1e of RT in packaging and
placement of tRNALy.3 •••••••••••••
1. 5. References •••••••••••••••••••••••••••••
Preface
42
44
86
..
ehapter 2: Ro1e of Pr160gag-po1 in Mediating the Selective
rncorporation of tRNALyo into Human rmmunodeficiency
virus Type 1 Partic1es ••••••••••••••••••••••••••
87
.
88
2.2. rntroduction •••••••••••••••••••••••••••
89
2.3. Materia1s and Methods ••••••••••••••••••
91
2.4. Resu1ts ••••••••••••••••••••••••••••••••
105
2.5. Discussion •••••••••••••••••••••••••••••
109
2.6. Acknowledgaments .•••••••...•••••••••••.
113
2.7. Refemces ••••••••••••••••••••••••••••••
114
2.1. Abstract
Preface
119
..
Chapter 3: Reverse Transcriptase Connection Domain Mutation.
in Pr160 gag-pol rnhibit the Select rncorporation of
•
Primer tRNALy.3 into HXV-1 ••••••••••••••••••
120
3.1. Abstract
..
121
3.2. Introduction •..••.••••.•.••••••••.••••
122
3.3. Materia1. and Method••••••••••••••••••
124
3.4. Resulta
..
143
3.5. Di.cussion
..
148
3.6. Acknowledgements ••••..••••••••••••••••
152
3.7. Ref.r.nc••.•..•.......•........•.......
153
xiv
•
Preface
160
..
Chapter 4: The Ganomic Placement of HIV-l Primer tRNALy.3
Oceurs in the Absence of a Punctional Protease or
IntegraB8 Sequences ...••••.•....•••....•..
161
4.1. Al:>stract •••••••••••••••••••••••••••••
162
4.2. Results and Discussion •••••••••••••••
168
4.3. References •••••••••••••••••••••••••••
172
Chapter 5: General Discussion
5.1. General Discussion •••••••••••••••••••
175
5.2. References
.
180
Original Knowledge •••
185
Chapter 6: Contribution to Original Knowledge
6.1. Contribution
t~
•
xv
•
List of Figures and Tables
P.g..
Chapter 1.
Figure 1. Organization of viral Sequences in vir.l DNA
and Genomic RNA ••••.•.•.•.••••••••••.•••.••••.
Figure 2. Schematic Diagram of The Matur.. HIV-l
Vi~ion
5
6
Figure 3. overview of The HIV-l Replication Cycle •..•.
B
Figure 4. Mechanism of R..troviral Revers.. Transcription.
9
Figure 5. Maturation of Human Immunodeficiency Virus
Typ.. 1 Viral Particl..s ••••••••••••••••••••••..
12
Retroviruses and Their Primer tRNAs •••••••••••
36
Table 1.
Chapter 2.
Figure 1. Structures of Wild-Type and Mut...'t HIV-l
Plasmids •••••••••••••••••••••••••••••••••••
95
Figure 2. 2-D PAGE Patterns of Low-Molecular-Weight Vir.l
RNA ••••••••••••••••••••••••••••••••••••••••
Table 1.
Percentag.. of
tRNA~
Isoacc..ptors in Total
Cellular and Viral tRNA •••••••••••••••••••••
Figure 3. Quantitation of
97
tRNAL~3
99
in viral RNA ••••••.•
100
Figure 4. Quantitative RT-PCR for Viral Ganomic RNA •••
102
Table 2.
Molecules of tRNALy.3 per Two Molecules of
Genomic RNA ••••••••••••••••••••••••••••..•••
104
Chapter 3.
Figure 1.
Schema~ic
Representations of wild Type and
Mutant Pr160 g • g -pol Pr..cursor Proteins •••••••
Table 1.
Mutations in HIV-l pr160 g • g -pol ••••••••••••••
Figure 2. 2-D PAGE Patterns of Low Mol ..culer
..
•
132
Characterization of Wild Typ" and Mutant HIV-l
Viral Partic188
Table 3.
131
w.. ight
viral RNA
Table 2.
129
..
135
Eff..cts of Mutations upon 2-D PAGE vir.l tRNA
Patterns and tRNALy.3 Incorpor.tion ••••••••
Figure 3. Quantit.tion of Genomic RNA in Viral RNA
xvi
136
•
Samples ••••••••••••••••••••••••••••••••••••
Figure 4. Quantitation of tRNALy.3 in viral RNA Samples
137
Figure 5. Western Analysis of Viral Proteins •••••••••
141
139
Chapter 4.
Figure 1. Schamatic Representations of wild Type and Mutant
HIV-l Viral DNA Sequences ••••••••••••••••••
Figure 2. In vitro Primer Extension Assay ••••••••••••
163
165
Figure 3. Quantitation and Primer Extension of Total Viral
RNA in Wild Type and Mutant Particles ••••••
•
xvii
167
•
List of Abbreviations
Ab
:Antibody
ASLV
:Avian Sarcoma L8Ukosis virus
AMV
:Avian Myeloblastosis Virus
CA
:Capsid
CMV
: Cytomegalovirus
env
:Envelope
Gag
:Group Antigen(Viral structural Protein precursor)
Gag-Pol
:Group Antigen-Polymerase Fusion protein(Viral
Enzymatic Protein Precursor)
HIV
:Human Immunodeficiency Virus
IN
: Integrase
LTR
:Long Terminal Repeat
MA
:Matrix
MoMuLV
:Moloney Murine Leukamia virus
Ne
:Nucleocapsid
PBS
:Primer Binding Site
P.R
:Protease
RSV
:Rous Sarcoma virus
RT
:Reverse Transcriptase
SDS-PAGE
:Sodium Dodecyl Sulfate-Polyacrylamide Gal
Electrophoresis
•
SIV
:simian Immunodeficiency virus
TCIDs.
:Dosage of 50% Infection of Tissue Culture Cella
2-D PAGE
:TwO Dimensional-Polyacrylamide Gal Electrophoresis
•
Chapter 1
Literature Review
•
l
•
1.1. Introduction
The discovery of a filterable,
transmissible agent associated with
avian leukosis sarcoma by Peyton Rous in 1911 is recognized as the first
landmark in modern retrovirology(2S4).
This
transmissible agent was
shown to have the ability to transform celis and to cause turnors, while
extracts of transplantable turnors in rats, mice, and dogs failed(254).
During the 19305, high frequencies of spontaneous leukernia were observed
in severai inbred mi ce strains.
lt was speculated that the etiological
agent of leukemia in mice and birds had a viral origin(92, 247).
It
WC1S
la ter shown that a significant portion of turnors is associated with RNA
tumor viruses(retroviruses), and that mast RNA tumor viruses capable of
transforming cells in culture are also defective for replication(3131.
Baltimore and Temin
independently pubUshed
protein reverse transcriptase (RT)
the
discovery of
in the spring of 1970(9, 292).
viral
RT is
a viral protein which has RNA-dependent and DNA-dependent DNA polymerase
activity, and it is capable of synthesizing viral DNA using viral RNA as
the template(10).
The discovery of RT challenged the central dogma of
unidirectional information transfer, fram DNA tn RNA ta protein(lO) .
In the early 1980s, Gallo and his colleagues isolated the first
human
retrovirus
established
from a
T-lymphoblastoid
cell
Une
fram cells of a patient diagnosed with
lymphoma(232,
233).
Later,
a
related
retrovirus
that
had
been
cutaneous T-cell
with
significant
differences in immunological cross-reactivity was isolated fram a T-cell
variant of a hairy cell leukemia cell line(148).
These viruses are now
known as human T-cell leukemia virus l(HTLV-l) and human
virus 2 (HTLV-2), respectively.
T-~ell
leukemia
Subsequent studies have shown that HTLV-
land HTLV-2 are the etiological agents of adult T-cell leukemia, and
atypical hairy-cell leukemia, respectively.
On Oecember 10, 1981, three independent studies were published in
The New England
Journ~l
of Medicine to describe a new class of acquired
immunodeficiency syndrome (AlOS)
drug users(10S,
199, 268).
among homosexual men and intravenous
In 1983, Moltcagnier and colleagues at the
Pasteur Institute in Paris reported the isolation of a T cell retrovirus
•
from a patient with l:nnphadenopathy, one of the early signs in patients
progressing toward AIOS(13, 282).
This virus was later shown to be the
2
•
etiological agent of AlOS. and it was renamed as human immunodeficiency
virus, HIV.
1.2. Retrovirus Classification
Based
on
the
pathogenici ty
of
the
viruses.
divided into three subfarnilies: 1) Oncovirinae.
oncogenic rnernbers of the retroviruses.
viruses.
retroviruses
are
These include aIl the
2) Lentivirinae,
or the -slow·
Members of this subfamily are characterized by their ability
ta cause slow progressive degenerative disease
Spumavirinae,
or the "foarny· viruses.
In cell culture,
effects
of
the
formation
vacuolation in infected calis.
3)
They are the least characterized
subfamily of the retroviruses.
with
in hast animaIs.
giant
they cause cytopathic
multi-nucleated
cells
and
Foamy viruses are alsa known ta induce
persistent infections without Any clinical disease.
Retroviruses are alsa categorized basad on their morphological
characteristics, and these different morphologies may reflect different
modes of viral assembly in vivo.
By virtue of their morphologies seen
with the electron microscope, retroviruses are classified into type-A,
type-B, type-Co and type-O particles(290) .
Type A particles form intracellular viral particles, and they have
not been shawn ta have any infectivity.
intracytoplasmic
forms
Thë;..re are intracisternal and
of type A particles.
Intracisternal
type A
particles are often found in cells producing type C or type 0 particles,
but their functions are not known.
Intracytoplasmic type A particles
have been shown ta be the precursor of sorne type B particles.
Type
B particles
morphological
features:
are
particles
which
1)
Particles
at
conta in
the
two
striking
plasma membrane show
doughnut shape(hollow-sphere in 3-D) and long spikes are seen at the
cell surface.
2)
The budded mature particles have electron-dense
nucleoids that are eccentrically located within the envelope particles.
Intracytoplasmic particles are found in the cytoplasm of the infected
cells. e.s. Mouse Mammary Turnor Virus(MMTV).
No
•
intracytopla"mic viral
structure
particle-producing cells until viral budding.
electron-dense
crescent-shaped(dome-shaped
3
can
be
found
in Type
C
During viral budding. an
in
3-D)
structure,
the
•
precursor of the: viral core,
plasma membrane.
is loacted on the cytoplasmic side of thlO>
Less prominent spikes than type B particles are tound
on the cell membrane surfaces where crescent-shaped(dome-shaped in 3-D)
structures are found.
electron-lucent
cores
Budded viral particles contain centrally locdted
in
immature
particles,
electron-dense cores in mature particles 1
e. s.
or
centrally
located
avian leukosis-sarcoma
Virus (ALSV) .
'IYPE: P particle3 lJav.a bath intracellular and extracellular forms
of viral particles, e.s. Mason-Pfizer Monkey Virus (M-PMV) .
particles,
Like type 8
they produce ring-shaped intracellular particles, and contain
eccentrically-located electron-dense nucleoid in mature extracellular
particles.
But unlike type B particles,
they contain shorter surface
spikes which are sirnilar ta type C particles.
1.3.1. Human
~unodeficiency
virus Structure and Genome Organization
HIV is a type-C-like retrovirus, and it belongs to the family of
Retroviridae and the subfamily of Lentivirinae.
Like all retroviruses,
HIV-l con tains of two copies of positive-sense single strand genomic
RNA, and each copy of the viral RNA is 9.2 kb in length(238).
genomic
RNA
encodes
viral
precursor
proteins (Pr5S gag
1
HIV-l
Pr160gag-pol.
envelope protein(gp160», viral regulatory proteins(Tat, Rev, and Nef),
and viral auxiliary pro teins (Vif, Vpr, and Vpu) (Figure 1 & 2). PrSS o• o
contains the sequences for matrix(MA), capsid(CA) , p2, nucleocapsid(NC),
pl and p6. In addition to MA, CA, p2, and NC, PrI600· o- pol contains the
viral enzymatic proteins(protease(PR), reverse transcriptase (RT), and
integrase(IN».
Envelope(Env) protein is made up of the gpl20 surface
protein(SU) and the gp41 transmembrane protein(TM).
HIV-I genomic RNA
is associated with the viral NC in the viral core along with the primer
tRNALys3, and the viral enzymatic proteins(PR, RT, and IN).
core is def ined as
the viral
The viral
structure enclosed by CA structural
proteins and components within (Figure 2).
Immediately outside of the
viral core is the MA, which is found underneath the viral envelope.
Like other members of the retrovirus family, HIV consists of a lipid-
•
containing envelope derived from the plasma membrane during budding .
Viral Env proteins are inserted into this membrane, and this membrane
4
.'
•
Proviral DNA
PBS
1
U3
~
gag
1 1
pol
~::;env :Pd~ï~_3~[E
Viral Genomic RNA
PBS
Ul
r ..v
env
o
TF1 PR
RT
IN
U3
Mature HIV-1 Virion
•
Legend
~ matrix
•
III capsid
cyclophilln. Vpr, or vn
o protease •
•
reverse transcriptase •
surface ~ transmembrane
~ primer tRNA
' \ genomic RNA
•
~ nucleocapsid
integrase
•
surrounds a viral structure composed of the MA proteins and the viral
core within(313).
Viral auxi1iary proteins(Vpr and Vif) and ce11u1ar-
derived protein cyclophilin A are founà ta associate with HIV-l virus,
but it is not c1ear if the subvira1 loca1ization of these proteins is
between the MA prote in and the viral core or inside of 'the viral core
structure (Figure 2).
Electron microscope studies estimate that most
retroviruses are rough1y spherica1 structures with a diameter between 80
to 130 nm(209).
re~rovirus
The 1ength of the envelope pro teins varies from one
ta the next, and theyare found on the surfaces of the viral
particles.
Envelope proteins are glycoproteins, and they contain type-
specifie epitopes.
These envelope glycoproteins are used to bind to the
target cell which the virus will infect.
1.3.2. OVerview of HIV-l Replication Cycle
Attachment of a retrovirus to its target cell requires specifie
i.nteraction of the viral Env gp120 SU protein and a cellular surface
receptor on the target cell(Figure 3).
Cellular surface receptor CD4
was the first identified receptor for a retrovirus, and the interaction
between CD4
After
the
receptor and the HIV-l Env has been intensely studied.
binding
of
the
retrovirus
protein
to
its
corresponding
receptor, receptor-mediated endocytosis(120, 198, 326), or fusion of the
viral envelope and cellular membrane(201, 279) (for review see (162)) is
required for the virus to gain access into the target cell.
For HIV,
after binding of the Env protein to CD4 receptor, the viral envelope and
the
cellular
cytoplasm(45,
proteins (1%
memorane
279),
fuse
to
release
the
the
core
into
the
and the envelope proteins and the majority of MA
of MA pro teins are associated wi th the viral core)
thought to remain at the plasma membrane (96) .
enters
viral
cytoplasm of
the
target cell,
Once
are
the viral core
viral encoded protein RT
initiates reverse transcription and converts viral genomic RNA into
~
double-stranded DNA.
The current model for the reverse transcription of genomic RNA
•
into double strand cDNA is shown in figure 4.
Synthesis of HIV-l viral
cDNA requires the usage of a packaged cellular tRNALys3 as the primer,
and the elucidation of the mechanism of selective tRNA packaging and
7
•
•
Overview of HIV-1 Replication Cycle
Adsoijltlon
r-
1..
Provirus
r::wrI
'0
--= """"""-
~
~..-.--
-=:..
' =- """"""Transcription
--
cDNA
.................................. /
,
~uclear
Localization
and Integration
~
~
.oo,:ranslatlon
tRNA 't
F
lcRNA
1 ' \Gag·Pol
Gag
Assembly
Maturation
Mechanlsm of Retrovlral Reverse TranscrlptJon 1
•
U3
A
U3
A
III
A' U5'ëIP
A US PBS
····-1-1- - - - - - - - - - - - - - - f l - H I J
A'US'
A US PBS
U3
9P
A
...·+-1==============::::t1=t:11-1.. . ,
...
1
PBS'
11
U3'
....
A' uS' I r
A US PBS
U3 A
...................................................
11
1
PBS'
PPT
...
1(
PBS'
•••
~
genomic RNA
ANase H degraded viral ANA
anllsense cDNA
sensecDNA
primer !RNA
U3
A US PBS
U3
U3'
U3'
A' US' 'iP
U3
A
1
PSS
)
11
U3'
A' US'
U3
AUS
A
A'
~;==================1==t+=I
U3' A' US' PBS'
U3' A'US'
•
J\
•
genomic placement have been the objectives of
HIV-I virus,
rny
studies.
Inside the
tRNALys3 is placed onto the primer binding site(PBS), a
region located very near the 5' end of the RNA genome(239).
reverse transcription,
During
viral protein RT binds to primer tRNALyo3 and
uses genomic RNA as the template for DNA polyrnerization(45).
Synthesis
of viral DNA begins with the synthesis of the minus-strand strong-stop
DNA(Figure 4).
Minus-strand strong-stop DNA synthesis initiates from
the 3' end of the primer tRNA, and is the first discrete intermediate in
retroviral DNA synthesis(step 1).
Minus-strand strong-stop DNA is then
translocated from its site of synthesis at the 5' end of the genomic RNA
to a
pos i tion on
the
3'
end of
the genomic
RNA R sequence via
complementary binding between the sense R sequence and the antisense R
sequence in the minus-strand strong-stop DNA(step 2).
transferring minus-strand strong-stop DNA from 5'
template is known as the first template switch.
synthesized by the RT,
This process of
to 3' end of the
As the viral DNA is
the RNA template is degraded by the RT-encoded
RNase H activi ty which 00150 facili tates the template swi tching (24) .
Minus-strand DNA synthesis continues until it advances past the plus
strand RNA PBS sequence(step 3 & 4), and it is followed by the synthesis
of plus-strand viral DNA.
The synthesis of plus-strand viral DNA is
primed by a stretch of RNA sequences, a poly purine tract(PPT) fragment,
near
the
3'
end
of
the
genome,
degradation' of the genomic RNA.
viral DNA yields
that
remains
.of ter
the
RNase
H
Continued synthesis of the plus-strand
the plus-strand strong-stop DNA,
and this strand
con tains a copy of sense PBS sequence that is dbrived from the 3' 18
nucleotides of the tRNA primer used(step 5).
The 3' end of the plus-
strand strong-stop DNA hybridizes to the complementary sequences at the
3' end of the elongated minus-strand DNA,
second temp1ate switch(step 6).
and this is known as the
DNA synthesis of both plus and minus
strand DNAs continues, and yields the full length proviral DNA(step
7) (for review see (291».
During the synthesis of the full length double-stranded HIV-1 DNA
sequences,
the cDNA becomes part of the pre-integration comp1ex(91).
Studies have shown that in Oncovirinae,
•
nuclear entry of the pre-
integration complex correlates with cell division,
membrane break-down(18l, 249).
Le. with nuclear
Lentivirinae, on the other hand, can
10
•
infect non-dividing cells, which appears to be due to the ability of the
pre- int.egration complex to be carried into the nucleus by 2 viral
proteins MA and Vpr(37,
119,
301).
The vltdl protein integrase is
responsible for viral DNA integration into the host cell genome. (32, 33,
74) .
The integrated proviral DNA is recognized as part of the host cell
sequence.
complex
The transcription of the HIV-l ge"ome is regulated by a
interplay between viral
regulatory proteins
and cellular
transcription factors
that interact with the viral LTR region(for
reviews see:
2531 i.
(6,
143,
trans-activator prote!n, Tat,
direct
interaction wi th
a
For example,
the expression of viral
enhances the viral RNA transcription via
viral
RNA sequence,
the
Tat
responsive
element(TAR) (83), and cellular transcription factors (88, 143, 153, 253).
The primary RNA transcript from the HIV-1 genome is 9.2 kb in
length, and this RNA is the source not only of genomic RNA in newly
produced virus and full length mRNA, but it also gives rise to smaller
mRNAs as a result of single and double splicing events.
During HIV-l
viral replication,
9.2 kb,
three major classes of viral RNA,
4.3
kb(singly spliced), and 1.8 kb(doubly spliced), are found in the viralinfected cells,
but only the 1. 8 kb class of viral RNA enters the
cytoplasm and is translated at the early stage of HIV replication(159).
The 1.8 kb class of viral RNA is known as the early class of viral RNA,
and it encodes viral regulatory proteins Tat, Rev, and Nef.
Tat enters
the nucleus, and it binds to the TAR sequence to enhance the elongation
of viral RNA.
Rev enters the nucleus to facilitate the transport of the
4.3 kb and 9.2 kb mRNAs from the nucleus to the cytoplasm.
The 4.3 kb
singly spliced mRNA encodes the Env protein and auxiliary proteins(Vif,
Vpr, Vpu) , while the 9.2 kb unspliced mRNA codes for the Gag and Gag-Pol
precursor proteins.
Expression of Rev allows the translocation of the
unspliced and the singly spliced viral mRNA from the nucleus to the
cytoplasmic domain of the infected cells by redirecting the viral RNAs
to a non-mRNA export pathway, such as is used for the export of 5S rRNA
and spliceosomal U snRNAs(84).
•
This Rev-mediated redirection of viral
mRNA export requires the interaction of Rev with the
Rev responsive
element(RRE) that is found in the 3' region of the Env-coding sequences
in the unspliced and singly spliced viral rnRNA.
11
The singly spliced and
'.
•
Maturation of Human Immunodeficlency Virus-1 Virai Particles
~I
Pr55geg
Pr1 &OlI8lIlIOl
gp160
gag
1
1
CVifl~~~=:I2~
pol
I!!I!!:Î
1
vpu
1
env
--
118 matrlx Il capsId lm nucleocapsid 0
;.:>:. ,:<,"',::,': ~ ;
..~..::,:~~.:.::.:~.:: !JI.
U////b
•
cyclophilin, Vpr, or Vd
o
trans/rame proteln
•
inlegrase
•
surface ~ transmembrane
..... primer tRNA
\
genomic ANA
~
maturation
0
protease •
p6
reverse transcriptase
•
unspliced viral rnRNAs are otherwise trapped inside of the nucleus in the
absence of Rev(75, 82, 109, 193, 194).
During viral assembly,
RNA.
primer
tRNA,
and
viral precursor proteins, viral genomic
sorne
other
virus
associated
materials
are
localized in close proximity on the cytoplasmic side of the plasma
membrane, anf. newly-formed immature viral particles then bud fram the
plasma membrane.
inunature virus
The probable arrangement of protein sequences in the
is sho\o/n in figure 5.
Processing of viral precursor
pro teins by viral protease transforms the
~lectron-lucent
structu~e
into the electron-dense viral core
viral core
of the mature virus(for
review see (212».
The conserved arder of
the viral structural protein sequences
found within the Gag precursor correspond to their conserved order
wi thin
the
virus
particles(319).
from
to
the
interior
of
the v.l.ral
The conservation of protein sequence order of Gag
precursor(MA,
CA,
retroviruses
implies
functional
the exterior
and NC)
domains
and Gag-Pol precursor(PR,
the
of
simultaneous
alignment
the precursor proteine
so
RT,
of
that
and IN)
the
in
various
each of
the
individual layers of the mature virus need not be assembled separately
and
sequentially(i.e.
sequences(MA/MA
and
sequences (PR/PR,
RT/RT,
the
CA/CA)
multimerization
and
dimerization
and IN/IN)
of
of
the
the
Gag
protein
Pol
protein
probably pre-exist in the form of
precursor protein) (26, 319) (Figure 5).
1.4. HIV-l viral A88embly: Viral Protain8, viral RNA, and Thair Rola8 in
Viral A88embly
The ultimate goal of viral infection is to release infectious
viral particles
from infected ceUs.
This requires that the viral
genome, viral proteins, primer tRNA, and cellular factors (synthesized by
separate mechanisms) be assembled in an orderly fashion.
The objective
of this work is to study the mechanism of primer tRNA incorporation into
HIV-1 and genomic placement of primer tRNALys3 during viral assembly.
•
Since viral proteins
and viral genomic RNA are
involved
in
these
processes, their mode of assembly into virus is relevant to our studies,
13
•
and below,
we examine in more detail the assembly of aIl
these viral
components ta produce a mature virus.
Our current scheme of viral assembly is largely based on a model
which was first proposed during the late 70·s(26).
This model proposed
the occurrence of the following events:
1.
Complexes containing viral envelope proteins migrate ta
the
cel1 surface and are inserted in the membrane at what will be site of
virus budding.
2.
Unprocessed viral precursor proteins are then transported ta
the budding site where they are found to be associated with viral
genomic RNA, primer tRNA, envelope proteins and other viral components.
3.
As viral maturation progresses,
viral
protease cleaves
the
precursor proteins to yield the viral structural proteins(MA, CA, and
NC) and the viral enzymatic proteins(PR, RT, and IN).
4. The respective virus proteins then associate with themselves
and/or with other pro teins to form the mature virus.
While a significant portion of the current assembly model remains
valid for
H~V
viral assembly, certain aspects of the model undoubtedly
require revision.
For example,
it has been suggested that Gag-Gag
interactions oceurs in the cytop!asrn te forro regular but electron-lucent
structures which condense only after transport to and interaction with
the cytoplasmic membrane (334) .
Also, envelope is made in the ER, and it
probably is carried to the plasma membrane in vesicles pinched off from
the Golgi.
Evidence exists for association of MA with Env(216), and
this may mean that the same vesicles carry Gag and Gag-Pol to the plasma
membrane as well.
These observations suggest that the interactions of
major viral components(Env, Gag and Gag-Pol) may occur in the cytoplasm
prior to the translocation of protein precursors to the budding site in
the plasma membrane.
1.4.1. Viral Proteine
•
Translation of the unspliced 9.2 kb HIV-1 mRNA yields the viral
pr..cursor proteins PrSS gag and Prl60gag-pol.
The PrSS gag encodes the
14
•
sequences of MA, CA, p2, Ne,
contains(in addition
pl. and p6 prot~insl while the Pr1600ag-pol
to MA,
CA,
p2,
and NC)
PR,
RT,
and IN.
The
conserved arder of the MA, CA, and Ne sequences within the Gag precursor
corresponds
exterior
to
to
precursor
interior
may
be
of
the
processing
virus(i.e.
their
arder wi thin
conserved
of
the virus (319).
important
for
precursor
the
the
This
rapid
proteins
to
virus,
goin9
arrangement
sequential
produce
fram
in
the
proteolytic
infectious
HIV-l
the multimerization of the Gag protein sequences(MA/MA and
CA/CA) and dimerization of the Pol protein sequences (PR/PR,
RT/RT, and
IN/IN) probably pre-exist in the form of precursor prote in) .
Since the expression of Pr5S 0ag precursor protein
alone
is
sufficient for the formation of virus-like particles, this property has
earned Gag the name of 'particle-making machine' (61, 319).
Mutational
analysis of Rous sarcoma virus(RSV) Gag suggests that there are three
assembly domains found in retroviral Gag precursor(3l9).
The spanning
regions of assembly domains in retroviruses do not directly correspond
to the mature proteins of the Gag sequence (MA, CA, & NC) (319) .
Assembly domain l(ADl)
is the membrane-binding domain,
which is
located at the N-terminus of the Gag precursor protein within the MA
sequence(18,
budding.
320,
336);
Assembly domain 2(AD2)
is required late in
Deletion of AD2 from the Gag precursor prevents the release of
budded viral particles to the cell surface(106) , and it is thought that
AD2 perhaps allows the particle to emerge from the 3urface of the cell
during budding(3l8).
For RSV, AD2 is located in p2b, which is found
between MA and CA(3l8), whereas for HIV-l, AD2 is in the p6 sequence
found at the carboxyl terminus of the Gag precursor(106).
chimeric
proteins
have shown
that
the
RSV AD2
and
Studies with
HIV-l
AD2
are
exchangeable and positionally independent(22l).
Assembly domain 3 (AD3)
is defined as
tight packing of Gag
the region contributing
molecules during viral assembly(18,
to
314).
the
Mutants
lacking the AD3
domain are unable to direct viral core assembly, and produce particles
of a low density.
Studies of the RSV/HIV chimeric protein expression
vectors show that ei ther of the
•
two copies of AD3
found
in the NC
sequence of HIV-l Gag can substitute for those in the RSV Gag(18).
It
is important to mention that the AD3 domains do not correspond to the
zinc-finger motifs in the NC sequence or signaIs known to be essential
15
•
for the packaging of genomic RNA,
because these
sign~ls
can be mutated
without affecting the packing of dense particles(103, 184).
1.4.1.1.1 Natrix
HIV-l MA sequence has at least three known functions.
anchors
PrSS gag
1)
It
to the plasma membrane during viral assembly through an
N-terminal myristylated amine acid glycine.
packaging of envelope proteins.
3)
2) It facilitates the viral
It helps to tlansport the pre-
integration cornplex into the nucleus in non-dividing cel1s.
The budding of the viral Gag particles requires the myristylation
of the MA sequence in the Gag precursor(34,
myristylation
of
the
Gag
MA
has
107,
been
219,
shown
242,
to
244), and
direct
the
intracytoplasmic transport of the Gag precursors te the plasma membrane
in at least one type 0 retrovirus, M-PMV(244).
Replacement of the MA
domain with a myristylation signal sequence is sufiicient for viral
particle formation(17S, 306, 320).
However, a large deletion in the MA
domain of the myristylated HIV-1 Gag (a type C retrovirus)
redirects
virus particle assembly from the plasma membrane to the endoplasmic
reticulum(80) or to the cytoplasm(27S).
Conversely, a single amine acid
mutation within the MA protein can signal a type 0 retrovirus, which
pre-assembles viral particles within the cytoplasm,
to follow a type C
retroviral particle assembly pathway, in which viral particles assemble
at the plasma membrane during the budding process(24S) .
Although myristylation app..ars to be required for the membrane
targeting of
important
the Gag
in
the
precursor,
intracellular
other
regions
transport
of
of
the MA are also
the
Gag
precursor.
Mutations in the MA protein of a myristylated Gag can cause a
accumulation
of
mutant
Gag
in
the
cytoplasm(334).
However,
large
co-
expression of this mutant with wild type Gag results in the packaging of
this mutant Gag into the viral particles.
This suggests that the mutant
Gag sequences must interact with the wild type Gag sequences in the
cytoplasm,
•
observation
and are then co- transported to the plasma membrane.
is
consistent with
the
hypothesis
that
interaction of Gag precursors occurs prior to the
sequences to the virus assembly site(334).
16
This
intermolecular
transport of Gag
•
Deletion and point mutations of the MA proteins have been shown ta
inhibit the packaging of Env protp.in(65,
87,
331), and Env protein
expression is crucial for the polarized budding of viral particles in
epithelial cells(186,
216).
In contrast to Bolognesi's model that
suggests Env and Gag pro teins are transported independently to the
plasma membrane(26),
these observations suggest that the interaction of
Env and Gag might occur prior to the Gag and the Env being transported
to the assembly site.
It is conceivable that efficient budding of viral
particles may require the cytoplasmic interactions of Gag-Gag and GagEnv sequences, and the role of Env protein in viral assembly will be
discussed in more details in a la ter section on envelope protein.
Alth~ugh
MA targets Gag to the plasma membrane at the late stage
of viral replication, MA can also target the pre-Integration complex
into the nucleus in the early stage of HIV-l infection.
Early studies
have shown that the MA protein can be found in the nuclei of an infected
cell(36).
Later it was found that a conserved basic sequence found in
MA is responsible for the presence of MA in nucleus, and that this basic
sequence functions as a nuclear localization signal (NLS) (37, 301).
The
MA NLS and the viral protein Vpr(which will be discussed in detail in
the Vpr section)
allow HIV-l virus to establish infection in non-
dividing cells by directing the migration of the pre-integration complex
into the target host cell.
During viral maturation,
a membrane-
associated tyrosine kinase phosphorylates approximately 1% of the MA
proteins at a carboxyl terminus tyrosine residue (97),
The tyrosine
phosphorylation of the MA pro teins triggers their incorporation into the
viral core, and the phosphorylated MA molecules bind to the IN sequences
in
the
mature
virus (98).
During viral
infection,
the
tyrosine
phosphorylated MA directs the migration of the viral pre-integration
complex into the nucleus (98) .
1.4.1.1.2. Capsid
Unlike the MA domain,
•
roles for the CA sequence other than its
structural one are not well defined.
However, this largest Gag cleavage
product contains an internal sequence of 20 amino acids(major homology
region, MHR), which is highly conserved in retrovirus Gag sequences(222,
17
•
319) .
The conserved nature of the MHR sequence implies an important
function of MHR in the retroviral life cycle.
sequences
N-terminal
to
the MHR,
Deletion of CA demain
or point mutations within
the MHR
sequence alter the morphology of the mature cone-shape core (63), and
mutations
in
the
MHR
regian
have
also
been
shown
efficiency of particle formation(51, 63, 277, 284).
to
reduce
Though the
the
~recise
raIes of MHR and CA sequences in viral assembly require further. study.
it appears that MHR mutations exert their effects by causing
of Gag/Gag or Gag/Gag-Pol
inte~actions
impa~rments
during viral assernbly and/or at
the later stage of viral core rnaturation(51, 63, 196, 277).
1.4.1.1.3. pZ
Proteolytic
processing
of
the
Gag
precursor
generates
the
structural proteins(MA, CA, and NC), p6, and two small polypeptides(p2
and pl) of unknown function.
p2 is a sequence of 15 amine acids found
between the CA and NC sequences of the HIV-1 Gag.
Limited information
is available for the role of the p2 domain in HIV-1 replication cycle.
An
in vitro proteolytic processing study has shown that the p2 domain of
HIV-1 serves as a negative regulator of
proteolytic site.
CA/p2,
p2/NC,
the cleavage of the CA/p2
There are five processing sites in HIV-1 Gag (MA/CA,
NC/p1, and p1/p6), and they are cleaved at rates which
differ by as much as 400 fold.
Removal of the p2 sequence or blocking
of the p2/NC cleavage site accelerates the rate of CA/p2 cleavage by 20
fold, implying that the processed intermediate C-terminal p2 tail lowers
the rate of the upstream CA/p2 cleavage during virus maturation.
Virus
produced from a p2 deletion mutant show wild type mature protein
sequences, but they are less infectious than the wild type virus.
It is
probable that p2 functions as a regulator for the proper assernbly of
virus by controlling the sequential processing of the precursor proteins
for the proper assernbly of virus during maturation(230).
1.4.1.1.4. Nuc1eocapaid
•
The HIV-1 NC protein has at least three known functions.
1) The
zinc fingers of the NC are involved in the packaging of the viral
18
•
genomic RNA.
2) The Ne facilitates genomic RNA dirnerization and genornic
RNA-primer tRNA annealing.
This genomic RNA-primer tRNA annealing
activity of NC will be further discussed in the primer tRNA section.
3)
The NC con tains AD3 domains that are involved in the tight packing of
the Gag molecules during viral assembly.
The zinc finger has been shawn to be involved in the recognition
of specifie nucleic acid sequences for a wide variety of eukaryotic
transcription factors(for review see (79».
In aIl known retroviruses,
the NC protein con tains either one or two copies of zinc finger(49, 121,
211) .
For example, while HIV and AMV have two zinc fingers in their
respective Ne
domain.
sequence,
MoMuLV has only one zinc
finger
in
its Ne
The RSV p12 sequence is one of the first NC proteins to be
linked to the genomic RNA dimerization and viral packaging(69, 205), and
similar results have been observed in ather retroviral systems (3, 103,
104,
204).
The interaction of NC protein with viral RNA has been
confirmed using in vitro binding assays(20, 22, 257).
of NC with viral RNA occurs via recognition of
The interaction
the RNA secondary
structure at the 5' end of the genomic RNA(see later section on genomic
RNA) (22, 44, 257).
It has also been suggested that NC promotes strand
exchange between double stranded and single stranded nucleic acids by
lowering the melting temperature of the nucleic acid duplex(295).
Mutagenesis studies show that when there are two zinc fingers, the
zinc
fingers
are
not
functionally
equivalent(30,
64,
102).
By
rearranging the locations of the first(proximal) and the second(distal)
zinc fingers,
replacing the proximal zinc finger wi th the distal zinc
finger or vice versa,
or adding an extra zinc
finger,
it has been
observed that the order and number of zinc fingers are less important
than the presence of two distinct fingers for genomic RNA packaging and
viral replication
in RSV(30).
AlI RSV mutants that have had
the
proximal and distal zinc fingers rearranged demonstrate only modest
reduction of viral infectivity.
identical
•
proximal
or
two
But RSV mutnts containing either two
id3ntical
distal
zinc
fingers
are
non
infectious.
RSV mutants containing two identical proximal zinc fingers
instead
the
of
efficiently.
wild
type
sequences
do
not
package
genomic
RNA
On the other hand, HIV-I mutants containing two identical
distal zinc fingers and HIV-I mutants containing the proximal and the
19
•
distal zinc fingers in reverse order are non-infectious. and
package viral genomic RNA.
unabl~
to
HIV-l mutants containing two proximo.l :oinc
fingers can efficiently package genomic RNA but show a reduced level of
viral infectivity(l02).
Retloviral genomic RNA is found as a dîmer inside the virus(the
mechanism of
RNA dirnerization will
be discussed
in
the genomic
RNA
section), and it has been suggested that viral genomic RNA dimerization
One in vitro study suggests
may be required for viral RNA packaging.
that only the basic ami no acid sequences f lanking the proximal HIV-l
zinc
finger
of
the
NC
are
important
dimerization(59).
while another report
finger
flanking
and
its
dimerization in vitro(55).
sequences
for
HIV-l
viral
RNA
finds both the proximal zinc
are
required
for
viral
RNA
While both of these studies suggest the
basic amine acid sequences found between the zinc fingers are important
for the HIV-l viral RNA dimerization in vitro, a new report shows that
substitution mutations of teis basic amine acid sequence result in the
production of non infectious viral particles that have wild type leveis
of genomic RNA packaging in vivo(2l4).
While this basic amine acids
sequence found in between the Ne zinc fingers is important to facilitate
the dimerization of viral RNAs in vitro.
genomic RNA packaging.
it is not important for the
This implies that either the in vivo and in
vitro viral RNA dimerization requirements are different, or that genomic
RNA dimerization is not a prerequisite for viral genumic RNA packaging
in vivo.
Unlike HIV-l and RSV, MoMuLV con tains only one copy of the zinc
finger.
zinc
The NC directed Mo-MuLV genomic RNA dimerization activities are
independent
in
vitro(235),
but
it
is
still
the
short
basic
sequences surrounding the zinc finger of NC that are critical for the
genomic RNA dimerization activity in vitro(58).
For MoMuLV. both the
zinc finger and the basic sequences that flank the zinc finger are
required for the efficient genomic RNA packaging in vivo(130, 241).
A chimeric MoMuLV proviral DNA was constructed,
in which
the
MoMuLV NC sequence in the MoMuLV proviral DNA is replaced by the HIV-l
NC
•
sequences.
Co-transfection
of
this
MoMuLV(HIV-l
NC)
and
an
untranslatable HIV-l proviral DNA into mammalian cells yielded mutant
MoMuLV viral particles that specifically package the HIV-l genomic RNA.
20
•
The reverse experiment was also done by replacing the HIV-l NC in HIV-l
proviral DNA wi th MoMuLV NC.
These studies demonstrated that the
encapsidation of the specifie viral genornic RNA is correlated with its
specific NC sequences (23) .
Similar conclusions were drawn in the RSV
system, i.e. the RSV NC is responsible for the selective packaging of
the RSV genomic RNA(68) .
Since NC has been shown to interact with RNA in vitro,
it was
proposed that the RNA molecules may act as a molecular scaffold to allow
the Gag pro teins to pack tightly together at the carboxyl ends via the
AD3 domains during viral assembly(18).
Cellular RNA may serve in place
of the genomic viral RNA packaging for dense viral particles that lack
viral genomic RNA(18), and significant amounts of cellular RNAs have
been
found
in both wild
type and viral
RNA packaging
deficient
mutants(103, 179, 180, 204).
By substituting the RSV AD3 domain in RSV NC with HIV-l NC
sequences,
it has been found that two small regions of the HIV-l NC
protein have the ability to tightly pack the RSV Gag molecules during
viral assembly(18).
Simultaneous substitution of functionally relevant
amine acids in both zinc fingers of HIV-l leads to a reduction of viral
protein export, indicating that the NC domain of the HIV-l Gag precursor
is also required for efficient assembly or release of the virus (64) .
This observation probably is the result of the disruption of the two
HIV-l NC AD3 domains.
A cross-linking study of the MoMuLV Gag suggests
that the zinc fingers within the viral particles are packed in close
proximity with the CA sequence, and these sequences are probably held in
close proximity within the immature virus by interactions between CA and
NC domains(llO).
The interaction of CA and NC during viral assembly is
further supported by an in vitro experiment(39).
In this experiment,
purified CA-NC proteins from RSV and HIV-l assemble only when CA and NC
are fused together, and the presence of viral RNA enhances the assembly
process.
This implies that an association between CA and NC during
viral assembly is likely to be important for a functional AD3 domain.
1 .•• 1.1.5. p6
•
21
•
p6 has at l.,ast two known functional
cycle.
They are:
roles in the HIV-l life
1) Th'. encapsidation of the auxiliary protein Vpr
into viral particles(see
~ection
on Vpr) (165), and 2) The
of viral particle r·,:'ease (lOG) .
ami no acid motif
An
enhancement
(LXX) 4 in the
carboxyl end of the p6 is found te be conserved in various strains of
the
HIV-l
sequences,
and
incorporation of Vpr(164,
1B7,
protein,
p6
i~
is
responsible
223).
At
for
the
the N-terminus of
viral
the p6
a PTAP sequence that is found in the praline rich region is
invo1ved
in
the
virus
assemb1y and
re1ease(131).
A proline-rich
assemb1y domain is a1so found in the RSV p2b sequence, the AD2 domain of
RSV(31B) .
A study of
chin.. rie
mutants
has
shown
functional1y equivalent to p2b of RSV(221).
that
p6
oC
HIV-1
is
Like the HIV-1 p6, RSV p2b
is required la te in budding, and it is thought that the AD2 found within
these sequences may be allowing the particle to emerge from the surface
of the cel1 during budding(31B).
Mutations in RSV p2b result in the
intracellular accumulation of the mutated Gag precursor, and a reduction
of viral partic1e re1ease(29).
It is interesting to note that p6 does
not seem to be important for viral particle production in the absence of
PR(127, 129, 145, 1B9, 255).
Inactivation of protease in the HIV-1 p6
mutant or RSV p2b mutant partially reverses
release. (131,
31B).
the defect
in particle
These observations suggest that the HIV-1 p6 and
RSV p2b are the AD2 domains in their respective retroviruses, and there
seems
to
be
a
functiona1
linkage
between
the
AD2s
and
their
proteases (31B).
1. 4.1. 2. Pr160""Il-pol
1.4.1.2.1. Expression
The order of mature proteins(MA,
CA,
NC,
PR,
RT and IN)
in the
retroviral Gag-Pol sequences is conserved in a1l known retroviruses.
The Gag-Pol fusion protein is typically expressed at 5-10% of the level
•
of
the
Gag
suppression
protein (137),
of
and
tr~nslation
expression
termination
of
of
Gag-Pol
Gag,
i.e.
requires
the
translation
continues beyond the termination codon for Gag(for a review see (177».
22
•
It is argued that this suppression provides at least two advantages for
the retroviruses:
synthesis.
1) It modulates the level of viral enzyrnatic protein
Presumably only catalytic arnounts of the viral enzymes are
required during the replication cycle in contrast to the higher levels
of structural proteins required for viral assernbly.
that
the
Gag
incorporation
moiety
of
the
of
the
Gag-Pol
Gag-Pol
by
2) . It is thought
precursor
drives
participating
in
the
the
virus
Gag-Gag
interactions during viral assernbly, thereby providing the inclusion of
viral enzymatic proteins into the virus.
There are two different mechanisms for retroviral suppression of
translational termination:
1) An in-frame read through suppression, and
2) A -1 ribosomal frameshifting (for review see (116».
For viruses
using in-frame read through suppression, Gag and Pol sequences are in
frame,
but separated by a translational terminational codon UAG.
A
small portion of the translational machinaries engaged in Gag protein
synthesis utilizes a tRNA to insert an amine acid in response to the UAG
termination codon.
Sorne mammalian type C retroviruses examined ta date
utilize in-frame readthrough suppression for their Gag-Pol synthesis.
Murine and Feline Leukemia virus are the examples
translational
readthrough
readthrough
signal
termination codon.
is
for
found
Gag-Pol
in
the
~f
retroviruses using
expression(170,
sequences
327).
The
downstream of
the
The signal is bipartite and consists of 1) An RNA
pseudoknot structure downstream of the termination codon,
and 2)
highly conserved purine-rich sequence found irnrnediately 3'
termination codon and 5' of the pseudoknot structure.
of
a
the
The pseudoknot
structure is defined as a stem-loop structure in which nucleotides in
the loop are paired with bases downstream from the stern to form a second
base-paired region(stem 2), and the two stems are stacked coaxially and
are connected by two loops(177, 23",
~63).
Another type of translational terrnination suppression is ribosornal
frameshi fting.
Sorne virus es (1. e.
HIV-l)
require one -1 ribosornal
frameshifting to express the viral Pol sequences,
resulting in Pol
codons placed in the -1 position relative to those for Gag.
•
Other
viruses(i.e. MMTV, BLV, and HTLV-l) require two frameshifts, where PR is
expressed as part of the Gag-PR following the first frameshift(PR codon
sequences are in the -1 position relative to the Gag codons) and RT-IN
23
•
sequences are expressed as part of
the Gag-PR-Pol resulting
from two
frameshifts(RT-IN codon sequences are in the -2 position relative to the
Gag codons).
Unlike readthrough suppression,
frameshifting occurs a
considerable distance upstream of the termination codon, and removal of
the termination codon does not alter the mechanism of fràmeshifting.
A
stable RNA stem-Ioop structure(and/or a possible pesudoknot structure)
3' of the frameshifting site and a hepatnucleotide signal immediately S'
of the frameshift site are important for the frameshifting events(40,
136) •
Currently there is no known retrovirus which uses +1 frameshifting
as its translational suppression mechanism for Gag-Pol fusion protein
expression, but the usage of +1 ribosomal frameshifting can be found in
a distant cousin of the retrovirus family,
the retrotansposon Ty1
element in the yeast, Saccharomyces cerevisiae(316) .
1.4.1.2.2. Enzymes coded ~ pr16ogag-pol
1.4.1.2.2.1. Protease
Protease is an 11 kDa viral prote in that can specifically process
the precursor pro teins into their mature forms(for review see (212».
HIV-1 protease is an aspartic acid protease(S6, 224), and activation of
the viral protease requires its dimerization(S6, 224).
that
the
maturation
proteolytic cleavage
of
the
virus
of
require
the
precursor
I t is likely
proteins and
the dimerization
of
the
the Gag-Pol
precursor in vivo.
Retroviral maturation is characterized by the transformation of an
electron··lucent viral core to an electron-dense viral core (lS,
328).
The condensed viral core is a rod-shaped structure in HTLV-l, but it has
a
cone-shaped structure
in
HIV-l.
Studies
have
sh'Jwn
that
the
maturation of viral particles correlates with the activation of the
viral protease(107, 154, 225).
It has been reported that the immature
l'etroviral core is stable under nonionic detergent conditions that will
•
lead to complete disruption of mature viral particles(226, 280, 329).
In avian retroviruses, viral protease is expressed in both Gag-PR
and Gag-Pol precursors (212) .
Mutational
24
analysis
shows
that
the
•
presence of functional PR in the Gag-PR precursor, but nct in the GagPol,
is
required
for
the
full
activation
of
the
Pol
sequences,
suggesting it is the PR on the Gag-PR that is responsible for viral
maturation(50, 281).
On the other hand, the viral PR sequences of MuLV
and HIV are derived from the Gag-Pol sequences, which are less abundant
in retroviral particles than the Gag-PR in avian retroviruses (212).
This implies that RSV may require more PR than HIV-l and MoMuLV for the
complete maturation of the viral particles.
[lrecursors
Digestion of the viral
fram an avian protease negative virus with exogenous
PR
yields complete and correct cleavage of the Gag precursor but incomplete
processing of Gag-Pol (280) .
Overexpression of
the
HIV-l Gag-Pol
polyprotein results in the cytoplasmic activation of the HIV-l protease
and inhibition of assembly and budding of virus-like particles (151) .
Together, these studies suggest the proper precursor protein folding and
quantity of PR are critical for the accurate maturation and budding of
viral particles during viral assembly.
Thus i t
appears that the
processing of the viral precursor proteins ta produce infectious viruses
is a highly regulated process(230).
It was thought that the proteolytic cleavage of precursor pro teins
in viral particles occurred only after the release of progeny virus from
cells(2l2), but a recent study suggests that efficient budding of HIV-l
virus
requires
the
initiation
of
proteolytic
cleavage
of
viral
precursors at the membrane of infected cells prior to the release of
virus(149) .
Partial inhibition of PR function yields aberrant, immature viral
particles(150).
Protease-defective virus encapsidate normal amounts of
genomic RNA(89,
90,
280),
but the thermal stability of dimeric RNA
genomes is altered(this will be discussed in more detail in the genomic
RNA section).
In HIV, we have found that inactivation of protease does
not affect selective tRNALys3 packaging and its genomic placement(to be
discussed in the later chapters).
1.4.1.2.2.2. Ravaraa Tranacriptaaa
•
HIV-l RT is a heterodimer, which consists of a 51 kDa subunit and
a 66 kDa subunit.
The dimerization of RT has been shown to be important
25
•
for its enzymatic activity(243) (for review see (270».
The HIV-1 p66 RT
subunit cantains 560 amine acids, and it is roughly divided into three
ragions,
the polymerase damain,
the
tether region,
and
the RNase H
domain.
The p51 subunit is derived from the same RT sequence as the
p66, and it differs from the p66 by 1acking the RNase H domain.
high
degree
of
amine
acid
homology
between
different
The
retroviral
polymerases and RNase H sequences suggests a high degree of structural
and
functional
sequences
of
retroviral
conservation(138).
the
tether
RTs (141).
region
By contrast,
are more
A1though
the
full
the size and
variable
among
polymerase
the
different
and
activi ties can be separated in other retroviral RTs (126,
RNase
169,
H
288),
there seems to be an interdependent relationship between the HIV-1 RT
polymerase domain and RNase H domain(124,
125, 234).
Data from X-ray
crysta110graphy studies have provided additional information about the
HIV-l RT structure.
right hand(163),
fingers,
palm,
The p66 subunit resembles the structure of a human
and the polymerase domain has been resolved into the
and thumb domains,
while the tether region has been
renamed the connection domain(163).
Various approaches have been taken to identify the primer tRNA
binding site on mature RT,
and the role of RT sequences
tRNA Lys3
be discussed
incorporation will
in
later
in primer
sections
of
this
chapter, and in la ter chapters.
1.4.1.2.2.3. Integrase
Integrase (IN)
is a
32
kDa
protein,
and mutations
sequence have been reported to alter viral assembly(78,
in
321).
the
IN
IN has
been shown to exist as a multimer either by a complementation assay(76,
298)
or through use of the yeast two-hybrid system(146).
Using the
yeast two-hybrid system, HIV-l IN has also been shown to interact with
Inil, a human homolog of yeast transcription factor SNF5(147), which ia
a transcriptional activator required for high level expression of many
genes.
•
promo tes
It
is
thought
integration
that Inil may encode a
and
targets
gen"s(147) .
26
incoming
nuclear
viral
DNA
factor
to
that
active
•
HIV-l
IN can be divided into three discrete regions:
terminal domain,
the core demain,
the N-
and the C-terminal damain.
The N-
terminal domain cantains a conserved zinc finger motif, which is found
in many
retroviral
and
retrotransposon
sequences(38,
141).
Point
mutAtions in the conserved zinc finger reduces the binding efficiency
between the IN sequence and the Inil protein in the yeast two-hybrid
system(147), as well as lessens the packaging efficiency of the Gag-Pol
precursor(78).
f inger and
However,
IN
removal of the second half of the IN zinc
sequences
downstream does
efficiency of the Gag-Pol precursor.
not af fect
the
packaging
The presence of an altered IN
sequence may interfere with the conformation of the molecule, and we
will show later that deleting the IN sequence in HIV-l Gag-Pol does not
affect its packaging.
The HIV-l
residues
IN core domain contains
(Asp64,
retroviral IN,
167, 287).
Aspl16,
and GlulS2),
three putative active-site
and
they are
retrotransposon IN, and sorne bacterial
conserved
in
transposa~es(a,
Mutation of any one of these three residues results in a
complete loss of in vitro integrase enzymatic activity(66, 77, 167, 168,
174, 297), and it blocks viral replication in cell culture(168,
267).
Point mutations in amine acid 136(Lys) and 138(Gluj in the core domain
in a
result
temperature
sensitive (TS)
mutant (321) .
Mutant
virus
collected from transfected cells grown either at 35°C or 39.S oC yields
viral particles with both wild type RT activity and protein patterns.
While wild type and mutant virus produced at 35°C possess infectivity,
the mutant virus produced at 39.S o C are capable of only one round of
integration,
although the resulting virus are produced at wild type
levels, and have wild type RT activity and protein patterns.
The fact
that these TS virus are able to integrate only once into the host cell
genome suggests
integration.
that
their defect is not directly related to viral
The defect responsible for the TS phenotype appears to
localized to a step post-integration but prior to particle maturation.
These
resul ts
suggest
that
the
core
domain
of
the
HIV-l
IN may
participate in viral assembly and/or virus maturation(321).
•
The C-terminal domain of HIV-l IN con tains a tryptophan residue
that
is
conserved
only
among
retroviral
IN,
retrotransposon IN or bacterial transposases(183).
27
but
not
found
in
Celetion of the C-
•
terminal domain has been found to lower the packaging efficiency of the
Gag-Pol sequences (78) , suggesting that the C-terminal IN sequences may
also have a rale in viral assembly.
1.4.1.3. Envelope
The HIV-I envelope protein, Env,
spliced mRNA.
that
is
is encoded by a 4.3 kb singly
Translation of this message produces a protein of 90 kDa
co-translationally
reticulum(ER) (218,
265) (for
glycosylated
review
see
in
the
(86».
endoplasmic
Additions
of
N-
asparagine-linked and mannose-rich oligosaccharide chains yield the 160
kDa Env glycoprotein precursor(gp160) (4).
Env precursor protein gp160
is cleaved by a cellular protease in the Golgi complex to yield mature
glycoproteins
gp120
and
gp41 (133).
The
cleavage
of
gp160
occurs
intracellularly via a cellular protease, and the processing is requirerl
for
th" intracellu1ar transport of the Env glycloproteins which are
necessary to produce infectious virus(317).
Externa1 viral gp120(SU) is
responsib1e for the binding to the target cell CD4 receptor(202), while
transmembrane gp41 (TM) anchors gp120 through noncovalent interactions
and mediates membrane fusion with target cells(28, 94, 95, 166, 272).
The synthesis,
modification,
and translocation of Env proteins
occur in the ER and the Golgi apparatus (218),
and they follow
the
cellular membrane protein transport pa thway , being transported to the
cell
surface
modification
proteins.
in
of
membrane
HIV-1
vesicles(for
glycoprotein may
review
require
see
(72».
cellular
The
chaperone
A recent study demonstrates that new1y synthesized HIV-l Env
protein associates
transiently with the cellular chaperone proteins
calnexin and calreticulin(213).
A1tered post-trans1ationa1 modification
of Env proteins has been shown to alter the migration of glycoprotein to
the
plasma membrane,
which markedly impaired the packaging of Env
protein into viral particles(108).
requirement
for
envelope
Oligomerization is another critical
protein
processing
and
its
cell
surface
expression, and the HIV-1 oligomeric Env glycoprotein is likely to exist
•
as either a
trimer or a
tetramer(70,
215,
262,
312).
Mutational
analysis studies suggest that the Env transmembrane domain is primarily
responsible for the oligomerization of Env protein(62,
28
71,
73,
296).
•
The oligomerization of Env protein oceurs prior ta the cleavage of Env
precursor
protein,
and
it
is
thought
that
the
oligomers
ramain
noncovalently attached post-clevage, and throughout the transport to the
membrane (62, 73, 296).
It has been established that retrovirus particle· formation may
occur independently of the expression of envelope proteins(19, 31, 218,
256) .
P~rticle
formation is thought to be driven mainly by the self-
association of Pr5S Qa g protein underneath the cel1 membrane, since other
viral gene products are dispensable for vir"l particle formation (99,
195).
However, an electron microscopy study has demonstrated structural
evidence of an interaction between Env and Gag protein during viral
assemblY(35), and deletion mutations in Gag can result in budded viral
particles
lacking Env protein(33l).
The HIV-1 Env proteins Most
probably interact with the Gag MA sequences via the gp41 cytoplasmic
tail.
Studies have shown that mutations in the gp41 cytoplasmic domain
lower the packaging efficiency of the Env proteins(67, 332).
In tissue culture cells, there is no obvious polarization of the
plasma membrane, but in epithelial ce1ls(such as those comprising the
lining of the vaginal and urethra1 tract, which are the target cel1s for
HIV-1 infection) membranes facing the lumen can be distinguished from
the baso1ateral membrane.
HIV shows po1arized budding in epithelial
cells, and it buds from the basolateral membrane.
While HIV-1 virus-
like particles can be released from the host cells in the absence of
HIV-l envelope protein expression(319) ,
proteins
the expression of envelope
is required to direct the polarized budding of HIV-1
epithelial cells(lS6, 216).
in
Other retroviruses have been shown to have
polarized budding in epithelial cells as well(15S, 252).
This coupled
with the fact that mutations in MA which prevent Gag/Env interaction
result in accumulation of Gag in cytoplasm, suggests that the envelope
sequences May interact with Gag Molecules in the cytoplasm prior to
reaching the virus assembly site at the plasma membrane,
and it is
thought that the intracytoplasmic domain of gp41 is involved in this
pro cess (lS6) .
•
These observations imply that there May be an Env
dependent regulated pathway for viral budding .
1 ••• 1 ••• Nef
29
•
Nef
protein
only encoded by primate
is
lentiviruses,
produced at aIl stages of viral gene expression.
and
is
Both Tat and Rev are
essential for HlV-1 gene expression, but rnRNA species encoding Tat and
Rev consist less than 20% of the doubly spliced 1. 8 kb early HlV-1
transcript population.
The majority of early viral rnRNAs encode Nef, a
27
that
kDa viral
assembly.
protein
Recent
may have an effect
reports
demonstrate
that
infectivity(42, 206, 276), but how is unclear.
during
Nef
HlV-1
viral
enhances
HIV-l
Virus produced from Nef-
negative mutants encapsidate wild type amounts of genomic RNA(2, 264),
and have identical viral protein patterns(264) as wild type.
type and Nef-negative virus have similar RT activity,
vitro endogenous RT and exogenous RT assays(2,
binding(4l, 264) and entry(2,
41)
41,
Both wild
using both in
264).
The viral
to the target cell, and the nuclear
import of the pre integrative viral DNA in infected cells are also
similar between the Nef-negative and wild type virus (264) .
Nef
may exert
its
effect
on viral
infectivity during
viral
assembly.
Nef is myristylated implicating its association with plasma
membrane.
A nonmyristylated form of Nef protein is as impaired as a
Nef-d<.leted virus.
Therefore,
Nef may exert its effect upon viral
infectivity through lts association with the plasma membrane.
One way
Nef can exert its effect of increasing infectivity is by modifying viral
components (i. e. Pr55 gag and pr160gag-pol) incorporated during viral
assembly.
For example,
it has been reported that Nef can bind to a
serine-specifie protein kinase present in many cell types (260) , and that
the phosphorylation of sorne of the MA serines residues during particle
formation is dependent upon the expression of the Nef gene product(Trono
et al, personal communication).
1.4.1.5. Vif
Vif,
like
Nef
protein,
particles(for review see (300».
associated with
•
system(185) .
can
alter
the
infectivity
of
viral
Vif is packaged into virus and it is
the viral core structures both in the HIV and SIV
lt is thought that Vif acts at the late stage of viral
assembly to allow the formation of particles competent for the early
30
•
steps of infection,
and Vif may enhance the proteolytic cleavage of
viral precursor proteins required for the appropriate packing of the
viral core during virus maturation (269).
The ratio of the amount of
Pr55 go g and processed Gag products found in wild type HIV is reversed in
a Vif-negative mutant, and is strikingly similar to the reverse ratio of
processed and unprocessed pro teins compared to wild type in proteasedefective Inutants(269).
Also, an observation has been made that Vif is
encoded by the Lentivirinae but not the Oncovirinae subfamily.
studies have
shown
that
in
HIV-1,
a member of
Recent
Lentivirinae,
the
precursor pro teins are processed prior to viral budding(149) , while the
processing of oncogenic type C retroviral precursor proteins occurs only
after the release of progeny virus from cells (212) .
It is suggested
that Vif may be involved in the proteolytic processing of precursor
proteins(269) , a pro cess that has been shown to be a highly regulated
and sequential process(230).
The function of Vif protein in viral infectivity is dependent on
the cell type producing the virus (93, 258, 302).
types (H9,
a
lymphocytic cell line,
lymphocytes),
Vif
is crucial
for
macrophage,
the viral
In restrictive cell
and peripheral blood
life cycle,
increase the viral infectivity by up to 1,000 fold(85,
160, 285).
contrast with the Nef gene, whose absence only reduces,
abolish viral infectivity,
eradicates viral
and it can
In
but does not
the absence of Vif protein in these viruses
infectivity.
Recent
reports
demonstrate
that
the
majority of Vif-negative mutant particles from restrictive cells have a
nonhomogeneous packing of the viral core,
and a
Vif-negative viral
population has a higher ratio of immature to mature virus than found in
wild type viral population(128).
In addition, Vif-negative virus from
restrictive cells also contain much reduced quantities of envelope
proteins
and
an
altered
ratio
of
unprocessed
to
processed
G~g
proteins(27, 269).
However, Vif-negative mutants demonstrate no sign of
viral
impairment when virus
replication
cells(COS, HeLa, and M8166 cells) (93, 258).
is produced
in permissive
This may be because the Vif
function is rescued by a cellular Vif homolog in permissive cells(294).
•
Immunofluorescence
studies
have
shown
that
Vif
exists
as
a
cytoplasmic prote in which exists in both soluble cytosolic form and
membrane-associated form
in
infected cells.
31
Vif
is
predominantly
•
present in the cytoplasm and closely colocalizes with the intermediate
filament vimentin.
Alteration of the vimentin filament structure with
drugs affects the localization of Vif, suggesting a close association of
Vif with
this
cytoskeletal
component(152).
The plasma membrane-
associated form of Vif is tightly associated with the cytoplasmic side
of the cell membranes(101).
The interactions between Vif protein and
cytoskeletal structure or cell membrane may assist the migration or
packaging of HIV-1 precursor proteins into newly formed viral particles
in an orderly fashion.
1.4.1.6. Vpr
Vpr was the first known virus-associated auxiliary protein (47.
333),
and it shares extensive homo1ogy with Vpr and Vpx of HIV-
2/SIV(293).
The viral encapsidation of Vpr allows lentiviruses to
replicate in non-dividing cells by facilitating the transport of the
pre-integration complex into the nucleus of non-dividing cells(119).
The incorporation of Vpr into HIV-1 viral particles requires the
expression of a
functional Pr55 gog precursor protein(172).
and
the
leucine triplet repeat sequence in the p6 region of the Gag protein is
responsible for this Vpr incorporation (164, 187, 223).
The C-terminal
region of Gag precursor in HIV-2 also appears responsible for
packaging of the Vpr homologue in this virus, Vpx(323).
the
By fusing the
HIV-1 p6 sequence to the C-terminus of the MoMuLV Gag precursor,
the
MoMuLV /HIV chimeric Gag protein can package the HIV-1 Vpr (that is
expressed in trans) inLû the chimeric retroviral particles.
Vpr would
otherwise would not be packaged into authentic MoMuLV viral partic1es.
This study demonstrates that the p6 domain of HIV-1 alone is sufficient
for the incorporation of Vpr into viral particles (165),
and the N-
terminal a1pha-helical structure in Vpr is involved in Vpr nuclear
localization and virus incorporation(60, 190, 191, 324).
It has been
suggested that HIV-1 Vpr is found primari1y colocalizing with the viral
core in the mature virus(307).
The homologue in SIV, Vpx has been shown
to be localized outside of the viral core in mature virus(330).
•
In addition to facilitating the transport of the pre-integration
complex inco the nucleus, Vpr has also been shawn to possess a second
32
•
function in HIV replication(48).
G2/M phase,
and as a
result,
Vpr induces cell cycle Arrest in the
Vpr prevents the establishment of a
chronically infected HIV-l producer cell line(250).
Independent studies
have shown that Vpr arrests the cell cycling in G2/M phase by inhibiting
the activation of p34 CdcL cyclin B complex(1l7, 144, 240), and the Cterminus
of
process (GO) .
Vpr
has
been
suggested
to
be
responsible
for
this
It has suggested that the Vpr-induced cycling Arrest
blocks the clonaI expansion of anti-HIV-specific T cells, dampening the
antiviral immune response(144), and that the Vpr-induced cycling arrest
may delay apoptosis of infected cells resulting in a hi9her quantity of
virus product'on from infected cells(ll7).
1.4.2. ViruB ABBociated RNA
1.4.2.1. GBnomic RNA
HIV-1 genomic viral RNA is a 9.2 kb rnRNA, which, like the cellular
mRNA,
contains a S'm7G cap structure and a poly A sequence at the 3'
terminus(4G).
Unlike cellular rnRNA, the nuclear export of the unspliced
viral mRNA requires the viral protein, Rev.
Expression of Rev allows
the translocation of the unspliced and the singly spliced viral rnRNA
from the nucleus to the cytoplasm of the infected cells (for reviews
see(52, 173»).
The specificity of the Rev response is conferred by a
highly structured RNA target sequence, the Rev response element(RRE),
which is
found within the HIV-1 envelope gene sequence(251).
displays a high affinity for Rev in vitro(53, 54, 118, 210, 335).
RRE
Rev
acts as a nuclear export signal that redirects RRE-containing viral RNAs
to a
non-rnRNA export pathway,
such as
the export pathway that is
involved in the transport of 5s rRNA and spliceosomal U snRNAs through
the nuclear pore(84).
Using the yeast two-hybrid system, a novel Rev
interacting protein, Rev/Rex activation domain-binding protein(Rab) , has
been identified(25).
The interactions of Rab, Rev protein, and the RRE
RNA sequences facilitate the transport of RRE containing RNA sequences
•
to the cytoplasm(25) , and Rev has been shawn to bind to a nucleoporinlike protein, Rip1p, in yeast(28G).
33
Whether the Rab is the mammalian
•
homo log of
the yeast nucleoporin-like protein (Riplp)
rel1\llins
to be
examined.
During
viral
assembly,
the
viral
genomic
RNA
is
selectively
packaged in ta viral particles despite the presence of a high cytoplasmic
background of cellular rnRNAs(for review see (lS.».
Genomic viral RNA
packaging requires the participation of tr4ns-acting Ne sequences, and
cis-acting packaging signals in the viral genome.
The role of
nucleocapsid sequence in RNA packaging was discussed in
el
the
previous
section, and the function of ais-acting elements involved in genomic RNA
dimerization and encapsidation will be emphasized in this section.
The packaging of retroviral RNA requires the presence of a
sequence in the genomic RNA.
mapped
to
the S'
end of
necrosis virus (SNV) (155,
The location of the
~
the RNA genomes of RSV,
266, 274, 283,
308).
~(200)
sequence has been
MuLV,
and spleen
The absence of these
~
sequences in the singly spliced and the doubly spliced viral RNA results
in
their
not
being packaged specifica11y into the viral particles.
Mutational analysis has s!lown that d .. letions
between
the
splice
donor
Rite and
the Gag
of HIV-1
start
RNA sequence
site
reduce
the
encapsidation efficiency of genomic RNA in ta the viral particles(3. 43,
176).
Chemical and enzyme probings reveal that the S'
region of the
HIV-1 RNA forms a defined and stable secondary structure(14, 114), and
similar observations have also been reported in Mason-Pfizer monkey
virus (113) .
Further in
~i
tro bindin,- studies wi th Pr55 0ao and HIV-1
genomic RNA suggests that the binding domain in the RNA genome includes
sequences
found
sequence(188).
from
the
splice
donor
site
into
the
Gag
coding
Similar results have been observed in MuLV(17).
The
binding domain in HIV includes three potential stem-loop structures in a
120 nucleotide segment flanking the Gag start codon(20), and these stemloops
have been shown
vitro(20, 257).
to bind strongly with
HIV-1
NC sequence
in
The US region of the viral RNA has also been shown to
have a role in genomic RNA encapsidation(299).
In an attempt to define the minimal sequence requirement for HIV-1
genomic RNA packaging, different fragments of HIV-1 RNA sequence were
•
fused with non-HIV-1 RNAs, and the abilities of these chimeric RNAs to
be specifically packaged into the virus were examined.
The resultll
obtained suggest that the HIV-1 packaging signal MaY be dispersed and
34
•
heavily context dependent(21).
Unlike MoMuLV(I) and RSV(161), for which
segments of viral RNA mediate efficient packaging of heterologous RNAs,
heterologous RNAs containing 5' regions of HIV-I RNA involved in genomic
RNA packaging are not efficiently packaged into the virus(21).
Although a single strand of retroviral RNA is sufficient for the
synthesis of viral DNA(142), viral RNAs are found in dimeric form in the
mature HIV-I virus.
maturation.
Dimerization of viral RNA occurs prior to virus
Studies of HIV-I and MuLV protease-negative mutants show
that viral RNAs exist as a less heat-stable dimer prior to protein
maturation, and these RNAs are being referred ta as the ·premature· RNA
dimer(S9, 90).
These observations suggest that the genomic RNAs in the
protease-negative immature virus assume a different conformation prior
to virus maturation.
It is not clear that dimerization of viral RNA is
a prerequisite for genomic RNA packaging, but it is interesting to note
that the location of the proposed dimer linkage sequences, sequences
that are believed to be involved in the initiation of RNA dimerization,
either overlap or are in close proximity with the '1' sequences in
different retroviral genomic RNAs.
In vitro studies have mapped an auto
complementary sequence (ACS) outside of the dimer linkage sequences in
the 5' leader region as part of the dimerization domain(197), and this
ACS domain is found in various strains of HIV-I(171,
MoMuLV(lOO) genome.
20S, 271), and
It is specu1ated that the ACS sequence forms a
hairpin that is the core dimerization domain of HIV-1 genome(171),
possib1y initiating dimerization by a loop-l.oop interaction involving
complementary base pairing between the two monomers(271).
Single base
mutations in the ACS sequence completely abolish dimerization in vitro,
while the introduction of compensatory(complementary) mutations restores
the process (217) .
This implies that the complementarity of the ACS
sequences is more important than the actual nucleotide sequences in ACS
for retroviral RNA dimerization.
•
1.'.2.2.1. aetrovira1 Primer tRN1•
35
•
Table 1. Retroviruses and Their Primer tRNAs 1
Retrovirus
Primer
Squirrel Monkey Retrovirus
tRNALysl.2
Caprine Arthritis Encephalitis Virus
Human Spumaretrovirus
Mason-Pfizer Monkey Virus
Simian Retrovirus 1,2(Type D)
Visna Lentivirus
Equine Infectious Anemia Virus
tRNALys3
Feline Immunodeficiency Virus
Human Immunodeficiency Virus 1,2
Mouse Mammary Tumor Virus
Simian Immunodeficiency Virus(mac)
tRNAPro
Baboon Endogenous Virus
Bovine Leukemia Virus
Feline Leukemia Virus
Gibbon Ape Leukemia Virus
Human T Lymphotropic Virus 1,2
Murine Leukemia Virus
Reticuloendotheliosis Virus
Simian Sarcoma Virus
Avian Sarcoma leukosis Viruses
tRNATrp
1Data was obtained from Leis et al. 1993, Regulation of Initation of Reverse Transcription
of Retroviruses. In A.M. Skalka and S.P. Goff(ed.), Reverse Transcription, Cold Spring
Harbor Laboratory Press, New York.
•
36
•
One of
the unique
features
of retroviruses
is
convert viral genomic RNA into double stranded cDNAs.
the ability ta
The retroviruses
use a cellular derived molecule, tRNA, as the primer to initiate this
reverse transcription process.
The inclusion of the cellular tRNAs
within the virus is a selective process (310),
and placement of the
primer onto the viral genome utilizes the 3'-terminal 18 nucleotides of
the primer tRNA which is complementary to a region near the 5' end of
the 355 RNA genome which is termed the primer binding site(PB5).
Different primer tRNAs are used by different retroviruses, and the
natural primer tRNAs discovered have thus far been confined to tRNAPro,
tRNATrp,
tRNALysl,2 and tRNALys3 (Table 1).
tRNAPro(lll, 228, 289) and
both tRNALys(227, 273, 309) isoacceptors are the primers for mammalian
retroviruses, while tRNATrp is the primer for aIl members of the avian
sarcoma and leukosis virus group examined to date(81, 112, 229, 261,
310, 311).
The PB5 sequence in human immunodeficiency virus type l(HIV-
1) suggests that the primer tRNA in this virus is also tRNALys3 (239),
one of the three major tRNALYs isoacceptors in mammalian cells(237).
It is not known whether tRNA Pro , tRNATrp, tRNALysl,2, and tRNALys3
share any common structural features which confer upon these tRNAs
properties required for them to act as primers.
For example, tRNAs are
known to be modified by cellular factors post-transcriptionally, and
these modifications are believed to be important for tRNA structurefunction
relationships(325).
No
common
post-transcriptional
modification has been found exclusively in retroviral primer tRNAs.
However, a specific modification of tRNATrp influences its ability to be
packaged into avian myeloblastosiD virus (156).
species
have
been
identified
in avian
cells,
'l'wo major tRNATrp
which differ
by a
methylation of nucleotide 7 in the amine acid acceptor stem: G or m2G.
However, only the non-methylated species is packaged and used as the
primer for avian retroviral reverse transcription.
to m2G may thus prevent
protein (s)
responsible
The methylation of G
the rer:ogni tion of tRNATrp by retroviral
for
the selective packaging
of
the primer
tRNATrp(156) .
•
In work to be described in a later chapter, we have estimated that
there are approximately 8 molecules of tRNALys3 found in each HIV-l
viral particle. The ratio of tRNALys3 to tRNALysl,2 molecules found in
37
•
the ":irus is simila,- ta the ratio of tRNALysJ ta tRNALys1,2 molecules
found in th.. cytoplasm,
suggesting that tRNALysl,2 and tRNALysJ have
similar viral packaging efficiency.
A separate report from our lab has
shown that the quantity of viral packaged tRNALysJ can be increased by
increasing the cytoplasmic concentration of tRNALysJ via expression of
the tRNALysJ gene (132) .
However,
the total amount of viral tRNALys
rnolecules remains at 20 molecules par viral particle,
suggestl~g
there
is a limit of the total number of tRNALys molecules that can be packaged
in the virus.
A major function of tRNA is ta contribute amino acids ta the
growing peptide chains(123).
tRNA is part of
prote in synthesis in mammalian cells (278) .
~
channeled cycle during
During protein synthesis,
the aminoacyl-tRNAs are directly transferred from the aminoacy1-tRNA
synthetase ta the elongation factor, eEFla, which carries tRNAs ta the
ribosomes, and
after peptide bond formation, the deacylated tRNAs are
transferred back ta their cognate synthetases for recharging with amino
acids.
The primer tRNA cannot be aminoacylated and still function as a
primer for RT.
is
In HIV-1 infected cells, no detectable cellular tRNALysJ
found deacylated,
completely deacylated.
while
the viral encapsidated primer
However,
tRNA is
it is not known if deacylation of
primer tRNA is a prerequisite for tRNA incorporation into virus, or if
tRNA deacylation occurs post packaging(132).
The cytoplasmic source of
primer tRNA is not known(whether it is aminoacylated or not), but it is
possible that the selective binding of a primer tRNA ta a viral protein
occurs during the translation when the protein and the tRNA are in close
proximity.
1.4.2.2.2. Interaction of primer tRNA~·3 and Genomic RNA
Although the PBS sequence on the genomic RNA is where primer tRNA
binds
and
initiates
reverse
transcription,
the
PBS
sequence
is
dispensable for selective tRNALys incorporation into the virus (140,
192).
•
Select primer tRNA packaging has been shown ta be independent of
genomic viral RNA encapsidation in MuLV(180), and we will show in this
thesis that HIV-l primer tRNALysJ packaging also occurs independently
from the packaging of HIV-l genomic RNA.
38
Mutational analysis has shawn
•
that the first nine SI nucleotides in the PBS sequence are important for
HIV-1
replication(246).
Transfection of mutant
HIV provira1
Dl'As
lacking the first 9 nucleotides of the PBS into COS cel1s yields non
infectious HIV-1 virus.
This implies that the annealing of the last 9
nucleotides of the primer tRNALys3 to the first 9 nucleotides of the PBS
could be important for the initiation of reverse transcription.
Further
studies have suggested that the first 6 nucleotides(304) of the HIV-1
PBS are sufficient for the production of infectious viral particles.
Mutant viral particles retaining only the first 6 nucleotides of the
wild
type
PBS
sequences
replicate with delayed
HIV-1
replication
kinetics, and eventually these viruses revert back ta the wild type PBS
sequences.
7his suggests the first 6 nucleotides of the PBS could be
the minimum requirement for the annealing of genomic RNA and primer tRNA
for viral replication(304).
Transfection of proviral DNAs with altered
PBS sequences(i.e. PBS sequences that are complementary with tRNAs other
than the natural primer, such as tRNAPhe, tRNAHis, tRNAIle, and tRNAser )
yield virus with slow replication kinetics (57,
these
viral
particles
containing
altered
182, 305,
PBS
315).
sequences
AU
initiaUy
demonstrate very slow replication kinetics, and they eventually revert
back
to
their
respective
replication rates.
wild
type
PBS
sequences
and
wild
type
The reversion to the wil.d type PBS sequence probably
resulted from the reverse transcription of the 3' 18 nucleotides of the
natural primer tRNA used for the synthesis of viral cDNA(see figure 4,
step 5).
HIV-1
Such experiments were performed with HIV-1,
RT and
the
genomic
RNA prefer
to
use
suggesting that
the authentic primer,
tRNALys3, for HIV-1 reverse transcription in vivo.
The.e results
are
found
even when
the
PBS
is
altered
to
be
complementary to a tRNA present in relatively high concentrations in the
virus.
Sequence comparison of the last 18 nucleotides of the tRNAs(the
PBS complementary sequence)
shows that tRNALysl,2 differs from tRNALys3
at 5 positions with the first
•
nucleotides
of
identical.
Like
10 nucleotides,
tRNALys3 and tRNALysl,2 (the first
t.RNALys3,
tRNALysl, 2
is
tRNALysl,2
and
tRNALys3
are
within
the last 3'
8 of
enriched
packaged into the HIV-1 viral particles (192) .
between
while
and
the PBS)
8
are
selectively
While the resemblance
the
suggested minimal
requirements of a functional HIV-1 PBS(304), tRNALysl,2 is nevertheless
39
•
nct used as the primer for HIV-l replication.
Transfection of HIV-l
proviral DNA containing tRNALysl.2 complementary PBS sequence does not
yield viral particles that use tRNALysl.2 as a stable primer(57, 182,
305), suggesting the complementarity of the genomic
R~~
PBS sequence and
the last 18 nucleotides of the primer tRNA is not sufficient for the
template/primer
interaction
in
This
vivo.
findings are further
supported by both in vitro(134, 135) and in vivo(303) primer/template
studies, which conclude that, in addition te the interaction between the
genomic RNA PBS and the
l~st
18 nucleotides of primer tRNA, an A rich
loop located upstream of the PBS has to interact with the anticodon loop
of the primer tRNA for reverse transcription to occur.
Enzymatic probing of the primer/template complEx suggest that the
methylated thio U at position 34 of the tRNALys3 anticodon loop is
involved in both primer/template and RT interactions in vitro(135),
suggesting the anticodon loop is important for HIV-l replication in
vivo.
The interaction between the tRNA anticodon loop and the genomic
RNA A-rich loop sequence, and the interaction between the 3' end 18
nucleotides of HIV-l primer tRNA and the genomic RNA PBS sequences both
are important for the HIV-l reverse transcription in vivo(134).
A
recent study has shown that is possible to crea te a mutant HIV-l virus
that uses tRNAHis as its stable primer by replacing both the A rich loop
and the PBS sequences in proviral DNA with sequences complementary to
both the anticodon loop and the 3'
respectively(303).
PBS is altered.
end 18 nucleotides of
tRNAHiD
tRNAHis is not used as a stable primer i f only the
This observation shows that the interaction between the
A rich loop of the genome and the anticodon loop of primer tRNA is
important for the HIV-l reverse transcription in vivo.
1.4.2.2.3. Interaction of primer
tRNA~·3
with Viral Protein.
Two mature viral proteins(NC and RT) have been shown to interact
with primer tRNALys3 in vitro(12).
annealing
of
tRNALys3
enta
in
The HIV-l NC protein facilitates the
vitro
transcribed
genomic
RNA
sequence(59) , probably by unwinding the secondary structure of tRNALys3
•
in vitro(157).
Similar observations have been made in the RSV and the
MoMuLV systems, where NC promotes the annealing of tRNATrp and tRNAPro
40
•
onto the PBSs of the RSV and MoMuLV genomic RNA respectively(236).
promotion of
The
tRNALys3 and genomic RNA annealing by Ne cceurs in a Ne
concentration depandant manner in vit:o.
the NC/ tRNALys3
A recent study indicates that
interaction is non-specif ic,
i. e.
resul ts from the
general ability of NC to interact with RNA(203). The in vivo role of NC
in the annealing of the tRNA Lys3 onto the genomic RNA is not known. The
removal of NC inhibits the viral packaging of the genomic RNA, making it
more difficult to evaluate the role of Ne in the annealing of the primer
tRNALys3 ante the genomic RNA in vivo.
It has been reported that the in
vitro annealing of tRNALys3 is independent of the presence of the two
zinc fingers,
but depends upon the presence of a basic amine acid
sequence between the two zinc fingers (55,
59).
A recent report has
shown that point mutations in these basic sequences results in non-
infectious viral particles with wild type level of genomic RNA(214).
These mutants may therefore provide a means to examine the role of Ne in
the annealing of the primer tRNALys3 onto genomic RNA in vivo.
tRNALys3 also interacts with RT.
Interaction of tRNALys and RT
induces conformational changes of RT(248), and an excess of tRNALys3
stimulates
the enzymatic activity of the p66/p66 RT homodimer(5).
However, an excess of primer tRNALys3 has also been sho'Nn to inhibit the
enzymatic activity of
the p66/p51 RT heterodimer,
suggesting
the
interaction of between the RT homodimer and tRNALys3 is different from
the interaction of RT heterodimer and the same tRNALys3.
It has been observed that the tRNA acceptor stem(which con tains
the sequences complementary to the PBS) is digested by RNase A only in
the presence of retroviral RT, suggesting a partial destablization of
this
region by the RT(259).
o:.uch destabilization might also be
important for the genomic placement of primer tRNALys3 in vivo. It has
been reported that the
T~e
loop, the D loop. and the anticodon loop of
the tRNALys3 interact with RT.
T~e
In vitro binding studies show that the
loop(322) and the D loop(259, 212) are partially protected from
nuclease digestion in a HIV-I RT-tRNALys3 complex.
In vitro binding
studies have shown that mature HIV-1 RT specifically interacts with
•
primer tRNALys3(11, 12), and the anticodon loop structure is involved in
this
process (11) .
The
importance
of
the
anticodon
loop
in
RT
interaction is further strengthened by a cross-1inking study between
41
•
tRNALys3
and RT(207).
In
this work,
four uridine residues
in
the
acceptor stem, the D loop, the anticodon stem, and the anticodon loop of
primer tRNALys3, respectively, were separately labeled with radioactive
4-thiouridine.
The labeled tRNA was
then incubated with mature RT
heterodimer and irradiated with UV-light
to crosslink the
protein.
only
Of
these
four
4-thiouridines,
the
tRNA and
residues
in
the
anticodon loop and acceptor stems are crosslinked with the mature RT,
suggesting the anticodon loop and acceptor stem are in close proximity
with RT heterodimer during the heterodimer RT-tRNA Lys3 interaction(207).
The
cross-linked
radioactive
labeled
viral
RT
was
further
fragmented by CNBr treatment, and the protein fragments were separated
by
polyacrylamide
gel
electrophoresis.
Sizing
analysis
of
the
radioactive RT fragments shows that regions of RT that interact with
tRNALys3 are within the C-terminal portion of the RT sequences (207).
One of these RT fragments contains the connection domain of the RT(207).
This is consistent with the RT crystal structure which suggests that the
position of pSl connection domain could be a candidate for tRNA-viral
RNA binding(163).
Although the connection domain varies in size and
sequences among different members of retroviruses, a tryptophan repeat
motif within the connection domain has been shown to be conserved in
various strains of HIV-l, HIV-2, and SIV(7).
in a
la ter chapter that alteration of
inhibits
the selective viral
We will provide evidence
the connection domain of RT
packaging of primer tRNALys3.
Another
fragment of RT that interacts with the tRNALys3 con tains the helix clamp
structure(which consists of the aH-turn-aI in HIV-l RT), and this helix
clamp
structure
enzyrnes(122).
aH
l,elix
is
found
in
several
nucleotide-polymerizing
A separate X-ray crystallography study suggests that the
structure
is
involved
in
DNA
binding(139).
Mutational
analysis has shown that the core of aH appears to play an important
role
in
template-primer
binding,
and
in
protein-protein
interactions(16).
1.4.2.2.4. Role of RT in packaging and placement of t~~·3
•
Since the
RT/tRNALys3
interaction is essential
for
initiating
reverse transcription, and because RT/tRNALys3 interaction has also been
42
•
observed in vitro, RT sequences are likely candidates for being involved
in
the
packaging,
tRNALyo3.
In
~he
and
possibly
the
genomic
placement,
of
primer
avian sarcoma virus, a mutant that lacks functional RT
activity does not selectively package tRNATrp into the virus, suggesting
the
RT
could
be
responsible
for
thi ..
process (229)
~
observation has a1so been reported in the MoMuLV system,
A similar
in which a
mutant virus that did not have any detectable RT activity did not
package MoMuLV primer tRNA, tRNAPro, efficiently.
This truncated MoMuLV
RT mutant lacks 40% of the RT sequence from the C-terminus, and results
from an insertion of 1 nucleotide in the RT sequences creating three new
pre~ature
termination codons in the RT(17S).
It is
i~teresting
that in
the avian virus both tRNATrp packaging and genomic olacement are reduced
when RT is removed.
On the other hand, in MoMuLV, the genomic placement
of primer tRNAPro is normal even though tRNAPro is not selectively
packaged and no RT activity is detected in MoMuLV.
This suggests that
in this virus RT MaY be involved in the selective packaging of tRNAPro,
but not in its genomic placement(179).
These difference might be
related to the structural differences found between avian and Murine
RTs.
For example, avian RT is a heterodimer, whereas the functional RT
OL MoMuLV is a monomer.
Avian RT has been shown to selectively interact
in vitro with tRNATrp(llS) ,
while MoMuLV RT do es not selectively
interact with tRNAPro(220).
In this thesis, we will demonstrate that the unprocessed Gag-Pol,
rather than the mature Pol sequence, is involved in the selective tRNA
incorporation
into
incorporation
of
the
the
HIV-l
primer
virus,
tRNA Lys3
suggesting
the
selective
into
may
occur via
HIV-l
interaction of primer tRNALys3 and the RT sequence within the Gag-Pol
precursor protein.
sequence
within
We will also show that the alterations of RT
Prl60gSg-pol,
more specifically,
mutations
in
the
connection domain of RT, inhibit selective tRNALys packaging.
While processing of the precursor pro teins is essential for the
maturation of viral particles, we will provide evidence to show that
maturation of HIV-l viruses is not required for the genomic placement of
•
primer tRNA, and a truncated Gag-Pol precursor that lacks the IN domain
can selectively package primer tRNALys3 and place the primer cnte the
HIV-l genome in vivo.
43
•
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•
85
Which
•
preface(Chapt~r
In
tRNALyo
this
1,0.': quantitatively dernonstrate
chapter,
incorporation
2)
into HIV-l by comparing
the
the
selective
concentrations
of
tRNALyn in low molecular weight cellular RNA and low rnolecular weight
viral RNA.
We then examined the viral factor.s required for selective
tRNALyu incorporation inta HIV-l hy addressing the following questions:
1) T5 the processing of Gag and Gag-Pol precursor proteins necessary for
the selecti,.. e
incorporation of viral
precursor pcotein
will
alon~
the expreesion of
sufficient
information
tRNA?
2}
If expression
is sufficient for
vir~l
Gag
alane
and Gag-Pol
for
selective
or Gag
particles
tRNA packaging?
Gag
format~~n.
alane
3)
0:
provide
Nill
the
removal of PBS sequences or inhibition of genomic RNA packaging affect
the selective incorporation of viral tRNA?
4) Since mature RT prote in
has been shown to specifically bind to primer tRNALys3 in vitro, will
the deleticn of RT sequence alter the selective packaging of tRNALys3
into the virus'",
5) How many primer tRNALys3 malecules are incarparated
inta the virus?
•
86
•
Ch&pter 2
The Role of Prl60gag-pol in Mediating the Selective
Incorporation of tRNALya into BIV-l Particles
•
87
•
2.1. Abatract
cos-7 cells transfected with HIV-l proviral DNA produce virus in
which three tRNA species are mast abundant in the viral tRNA population.
Theze
tRNAs have been identified through RNA sequencing
tRNALy"3,
the
isoacceptor
primer
family.
tRNA
in HIV-l,
These RNAs
and
members
of
60% of
the
represent
techniques as
the
tRNALY"1.2
low rnolecular
weight RNA isolated from virus particles, while they represent only 6%
of the low molecular weight RNA isolated from the COS cell cytoplasm.
Thus tRNALyn is se!ectively incorporated inta HIV-l particles.
measured the
We have
ra tio of tRNALys3 molecules to copies of genomic RNA in
viral RNA samples. and have calculated that HIV-l con tains approximately
8
molecules
tRNALys3/2
copies
genomic
RNA.
We
have
aiso
obtained
evidence that the Pr160 0 3.g-pol precursor is involved in primer tRNALys3
incorporation into virus.
First,
selective tRNALys
incorporation and
wild type amounts of tRNALys3 were maintained in a protease-negative
virus unable to process the PrSS gag and Pr160gag-pol precursors,
indicating that precursor processing was not required for primer tRNA
incorporation.
Second, viral particles containing only unprocessed
gag
PrSS
protein do not selectively incorpora te tRNALYs, while virus
containing both unprocessed PrSS gag and Pr160gag-pol proteins demonstrate
selective
tRNALys packaging.
Third,
studies with a
proviral mutant
containing a deletion of most of the reverse transcriptase sequences and
approximately
113
of
the
precursor resulted in the
integrase
1055
sequences
in
the
Pr160g a g-pol
of selective tRNA incorporation, and an 8
fold decrease in the amount of tRNALys3/2 copies genomic RNA.
We have
also confirmed herein a previous study which indicated that the primer
binding site is not required for the selective incorporation of tRNALys .
•
88
•
2.2. Introduction
A limited nurnber of tRNAs derived fram the infected hast cell
packaged into retrovirus during virus assembly (9, Il, 24. 31, 33).
ctr~
One of
these i5 termed the primer tRNA because it 15 used to prime the reverse
transcriptase(RT)-~atalyzed synthesis
of
retroviral
strand DNA.
minu~
AlI rnembers of the avian sarcorna and leukosis virus group examined ta
date
use
tRNATrp
(10,14,26,33,39,40),
(13,24.37).
as
primer
for
reverse
transcript ion
whereas the murine leukemia virus employs tRNAho
and mouse rnanunary
tumar
tRNA Lys3
virus utilizes
(25,38).
The J'-terminal 18 nucleotides of primer tRNA are complementary ta
regian
near
site(PBS).
tRNA
in
the
5'
end
of
the
RNA,
termed
the
primer
binding
The sequence of this site in HIV-l suggests that the primer
this virus
is also
tRNALj'o3,
isoacceptors in manunalian cells
either
355
d
a
-free-
(8,10,11,38,39).
state
or
(28,29).
the
three major
tRN~Lj'u
Retroviral tRNA is found in
associated
with
the
viral
genome
When the viral 70S RNA complex is used ta direct DNA
synthesis cata1yzed by RT
associated
one of
than
the
(11,18,24,37,39),
one of
others,
ln HIV-l,
serves
these bound tRNAs,
as
a
primer
for
more
tightly
DNA synthesis
the tightly associated tRNA
is
tRNALya3
(16) .
During viral assembly,
tRNA packaging is selective,
and in HIV-l
produced by transfecting COS cells with HIV-1 proviral DNA,
isoacceptors,
packaged (16),
isoacceptor
tRNALyol.2 and tRNALyo3.
the tRNALyo
are the major abundance tRNAs
These tRNAs are selected from over 100 different tRNA
species
in
mechanism responsible
the
for
cytoplasm.
and we are
this process.
investigating
Previous work has
the
indicated
that the reduction or absence of viral genomic RNA incorporation does
not
affect
primer
retroviruses
are
tRNA
incorporation
into
virions(20,26).
PrSS gag and Pr160gag-pol precursors
assernbled using
which are not c1eaved until after viral budding
that
the selection of
•
sequences
processed
for
and
Pr160 gag -pol
NC and RT proteins,
have
it is likely
PrSS gag cantains the amina acid sequence for
prote in (NC),
forms)
(17,41),
tRNALyo may occur by the binding of tRNALya to
either of these proteins.
nucleocapsid
Because
been
shown
89
conta in
Both Ne and RT
to
bind
to
the amino acid
1in their fully
tRNALya3
in
vitro
•
(1,27.32).
the
The absence of RT protein has a1so been reported to affect
incorporation of primer tRNA in both murine
(21)
and avian
(26)
retrovirus.
In
this
report,
we deterrnine
the
incorporated into wild type HIV-1, and
binding
site(PBS),
selective
viral
incorporation
protease,
of
tRNALys
number
of
tRNALYs
molecules
examine the role of the primer
PrSS gsg •
and Pr160gsg-po1 on the
into viral particles.
This
is
accomplished through the analysis of the tRNA population in wild type
and mutant HIV-l particles produced by transfecting COS-7 cells with
wild type and mutant HIV-l pr"viral DNA.
supporting evidence for the involvement of
tRNALys packaging .
•
90
The data obtained provide
uncleaved Pr160 gs g-po1 in
•
2.3. Materials and Methods
A schema tic representation of the plasmids
Plasmid construction.
used for the transfections carried out in this study are shown in Figure
1.
SVC21 BH10 contains wi1d type HIV-l proviral DNA sequence.
SVC2l
P(-) differs from SVC21. BHlO by a single point mutation at position 25
of
the protease region,
converting Asp25
ta Arg25.
Transfection of
SVC21 P(-) produces noninfectious viral particles containing wild type
genomic RNA and unprocessed precursor proteins PrSSoao and Pr1600ag-pol
(12).
Both
SVC21
BH10
and
SVC21
P(-)
are
gifts
from
E.
Cohen,
University of Montreal.
SVC21 MSCI contains an in-frame deletion of RT and approximately
1/3 of
integrase,
and was derived from SVC21 BHlO.
completely digested with
Biolab).
and
deleted
a
fragment
restriction
enzyme MSc1
(New England
The resulting 12 kb fragment, containing the plasmid vector
H:i.V-l
electrophoresis,
using
the
SVC21 BH10 was
was
fractionated
using
and after extraction from the gel,
mini-spin
was
genome,
""luron,
self-1igated
and pheno1-ch10roforrn
with
T4
DNA
ligase
agarose
further purified
extraction.
The
(Pharmacia).
DNA
pSVGAG-RRE-R
and
sequencing was perforrned to confirrn the deletion.
The
construction
and
characterization
of
pSVGAGPOL-RRE-R and pSVGAGPOL-RRE-R P(-) were as previously described
(34,35).
Viral
production
from
these
two
plasmids
transfection with the REV protein expression vector,
requires
pCMV-rev.
coCo-
transfection of pSVGAG-RRE-R with pCMV-rev produces virus-like particles
containing unprocessed PrSS gag precursor prote in, while co-transfection
of pSVGAGPOL-RRE-R P (-)
with pCMV-rev produces virus-like particles
containing both unprocessed PrSS lIAlI and
Pr1601lAlI-pol.
AlI restriction and modification enzymes are used according to the
specifications of the manufacturer.
Production of wild type and mutant BrV-1 virus.
Transfection of
COS-7 cells with wi1d type and mutant proviral DNAs using the calcium
•
phosphate method was as previously described (17).
from
the
cell
culture
medium
63
hours
Virus was isolated
post-transfection.
The
supernatant was first centrifuged in a Beckman GS-6R rotor at 3000 rpm
91
•
for
30
minutes,
t~ie
and
virus was
then pelleted from the resulting
supernatant by centrifuging in a Beckman Ti45 rotor at 35,000 rpm for
one hour.
The viral pellet was then purified by centrifugation at
26,500 rpm for l hour through
l5~
sucrose cnte a
sucrose cushion,
65~
using a Beckman SW4l rotor.
Isolation of viral RNA, and human placental tRNALyo isoacceptors.
Total viral RNA was extracted from viral pellets using the guanidinium
isothiocyanate procedure (4).
The purification of tRNALys3 and tRNALysl,2 from human placenta was
performed as previously described (16).
The term tRNALysl,2 refers to a
population of two tRNALys species which differ by one base pair in the
anticodon stem (28).
In this paper, spots 1 and 2 are collectively
referred to as tRNALysl,2 since we have not sufficiently characteraed
these two species to distinguish tRNALysl from tRNALys2 (16).
RNA labeling.
The fractionated RNA samples were labeled using the
32 pCp 3' end-labeling technique (3).
32pCp was made as follows: 5 mCi
32
of garnma- P-ATP (specific activity 3000 Ci/mmole, Dupont, Canada) was
dried down in a microcentrifuge tube using N2 .
100 ul of the following
reaction solution was added (reaction solution: 50 mM Tris-HCl, pH 9.2,
5 mM MgC12' 3 mM di thiothrei tol, 5~ bovine serum albumin, 1 uM 3'cytidine monophosphate,
and 10 units T4 kinase).
The reaction was
incubated at 37°c for 3 hours, and the conversion of 3'-CMP to 32pCp was
monitored using PEI thin layer chromatography in 0.8 M NH 2S0 4 , which
separa tes 32pCp from AT32p.
Labeling of the RNA with 32pCp was as previously described (3,18).
After labeling, free 32pCp was removed from the labeled macromolecules
either
using
G-50
Sephadex
(Pharmacia)
home-made
spin
columns,
equilibrated with TE buffers (10 mM Tris, 7.5; 1 mM EDTA) , or during the
electrophoresis run.
Before analysis by polyacrylamide electrophoresis,
the samples were heated at 90°C for 2 minutes.
One- and two-dimensional polyacrylamide gel electrophoresis (lDPAGE and 2D-PAGE).
Electrophoresis of viral RNA was carried out at 4°C
using the Hoeffer SE620 gel electrophoresis apparatus.
•
cm x 32 cm.
The first dimension was run in a
10~
Gel size was 14
polyacrylamide/7 M
urea gel for approximately 16 hours at 800 volts, until the bromephenol
blue dye was beginning to elute from the bot tom of the gel.
92
After
•
autoradiography,
the
piece
of
gel
c:"ntaining
RNA was
eut
out
and
embedded in a second gel (20% polyacrylamide/7 M urea) and run for 30
hours
(25
watt
limiting),
followed
by
autoradiography.
All
electrophoret:c runs were carried out in 0.5 x TBE (1 X TBE = 100 mM
Tris; l mM borie acid; 2 mM EDTA-Na2)' The electrophoretic gel patterns
shown in this paper show only low molecular weight RNA, since the high
molecular weight viral genomic RNA cannot enter the polyacrylamide gels.
Furthermore,
these patterns represent only the most abundant
tRNA
species present, since the high specifie activities of the labeled tRNAs
used
will
reveal
more
minor
aboJndance
species
with
longer
film
exposures.
The signal intensity of each radioactive low molecular weight RNA
species in the 2D-PAGE RNA pattern was determined using phosphor-imaging
(BioRad, Toronto, Ont.).
Measurement of tRNALyo3 using RNA-DNA hybridization.
amount of tRNALys3 in viral RNA,
To measure the
we have synthesized an 18-mer DNA
oligonucleotide complementary to the 3' 18 nucleotides of tRNALys3 - (5'
UGGCGCCCGAACAGGGAC 3'). This probe hybridizes specifically with tRNALys3
(16,17),
and was hybridized to dot blots on Hybond N (Amersham) of
either purified human placental tRNALys3 or total RNA from wild type and
mutant virus.
The DNA oligomer was first 5' -end labeled using T4
polynucleotide kinase and gamma- 32 P-ATP (3000 Ci/mMol, Dupont Canada),
and specifie activities lOS to 10 9 cpm/ug were generally reached.
Approximately
10 7
cpm oligomer was
generally
used
per
blot
in
hybridization reactions.
Me&surement of viral genomic RNA using quantitative peRo
Labeling of primer: l nmol of sense primer is mixed with 50 uci of
32P-gamma-ATP (Dupont), and end labeled with 3 ul of T4 PNK (10 U/ul,
Pharmacia) in kinase buffer (70 mM Tris HCl (pH 7.5), 100 mM MgC1 2 , and
50 mM DTT) at 37°C for l hour.
Free radioactive label is removed by
passing through a G2s spin column.
Reverse transcription: 10 pmol of cold antisense primer is mixed
with a known volume of viral RNA or in vitro-transcribed RNA in a total
•
volume of 16.5 ul. Three drops of oil layer is placed on top of the
primer/template
mix
to
avoid
evaporation.
Tubes
containing
primer/template mix are heated at 85°C for 3 min and they are slowly
93
•
cooled down to room temperature to allow primer/template annealing.
8.5
ul of RT master mix (30 mM Tris-HC1, pH 8.3, 5 mM MgC1 2 , 6 mM DTT, 1.5
uM dNTPs, 0.5 ug BSA, 10 units RNase inhibitor (Promega), and 5 units
MMLV RT (Pharmacia»
is added to the primer/template mix.
tubes are incubated at 37°C for 1 hour.
Reaction
is terminated by
RT activity
incubation at 95°C for 5 minutes.
Polymsrass chain rsaction:
75 ul of PCR masteL mix (10 ul 10X Taq
DNA polymerase buffer (BRL/GIBCO), 10 ul 2 mM dNTPs,
l ul 32p labeled
primer mix, and 1.25 units of Taq DNA polymerase (BRL/GIBCO»
is added
to the reaction tubes containing reverse transcription products which
are incubated at 95°c.
amplification
elongation,
Reaction tubes are subjected to 19 cycles of
(denatu:c:ation,
72°C,
3 min).
94°C,
1 min; annealing GOoe,
1 min; and
Aliquots of PCR products are run on a 8%
polyacrylamide gel with 7 M urea to separate amplification products from
excess radioactive primers.
The gel is dried at
soce
for 2 hours, and
then exposed to a phosphor-imaging screen for quantitative measurement
on a phosphor-imager.
Quantitativs dstsrmination
viral
~
antisense
samplss:
primer
amplification.
o~
gsnomic
Sense primer (JS1)
(JAl)
~
in wild typs and mutant
50 ATTCGGTTMGGCCAGGGGG3 0 and
50 GGGATGGTTGTAGCTGTCCC3'
A 148 bp
fragment,
corresponding
are
used
for
to HIV-l
PCR
3B DNA
sequence 843-990, ic amplified.
A standard curve is established by using known amounts of in vitro
transcribed RNA for RT-PCR reaction.
A 957 base RNA fragment (sense) is
made from a linearized DNA plasmid, pEA2, with T7 RNA polymerase (pEA2
plasmid is kind gift from E.J. Arts of the McGill AIDS Centre).
This
RNA fragment corresponds to HIV-l 3B DNA sequence 473-1420.
Quantitation
of genomic
RNA
in wild
type,
mutant viral RNA
samples, and in vitro transcript RNA are done by phosphor-imaging (BioRad).
The relative intensity of amplified signals are used to determine
the concentration of genomic RNA in the viral samples using the standard
curve .
•
94
Figure 1. Structure of wi1d type and mutant HIV-1 p1asmids.
(B) SVC21 BH10 p1asmid contains the wi1d type HIV-1 provira1
DNA sequence. (C) SVC21 P(-) contains a protease-deficient
HIV-1 provira1 DNA due to a point mutation at amino acid 25 in
the protease region (12).
(D)SVC21 MSC1 is an in-frame RT
de1etion mutant derived from SVC21 BH10 as described in
Materia1s and Methods. The dashed 1ine of SVC21 MSC1 indicates
the de1etion region.
(E)pSVGAG-RRE-R and (F)pSVGAGPOL-RRE-R
P(-), which serve as temp1ates for the synthesis of either
Pr55ga g or unprocessed Pr160ga g-pol, respectively, were
constructed as has been previously de~cribed (32,33).
•
•
•
•
~::;
B. lI1IC21 BRl.
1
U3
C.
5...
~
gag
pol
""".hg"
lI1IC21 P(-)
~
U3
5...
1 1
gag
1
LI
pol
'Yffurvpu
-
:Ff-J 031fJ
1
~~:~ OllE
I:œ!:.I
'i'fh
'0'
... ,
vpu
D, SVC21 MSC:r
PBS
1 U3
~I.lJ
MSCI
gag
1
Jpro
11
-
~,1 int r wl:œ!:.Ir~i:';
:p~
vpu
_net
env
E, pSVGAG-RRE-R
Sac l
!J.-------,g==a:-=g;----.,\ PPE 1
F, pSVGAGPOL-RRE-R P(-)
lUIp~5-Gly~5
Sac l
li
gag
1
Ll
Sal l
pol
ryn!1 ME
1
03
1
~
Figure 2. Two-dimensional PAGE patterns of low molecular
weight viral RNA.
Electrophoretic conditions are as
described in the text .. A, uninfected COS cell; B, wild
type virus-SVC21 BHI0; C-F, mutant viral particles: C,
SVC21 P(-); D, SVC21 MSC1; E, pSVGAG-RRE-R; and F,
pSVGAGPOL-RRE-R P(-).
fi
•
•
..
•
ü
w
..
ft
•
,,
.. ~it-:
•
c
c(
98
•
•
Table 1. Percentage of tRNALys isoacceptors in total cellular and viral tRNA *
tRNA source
A. COSCELL
%tRNALysl,2
(spot 1)\1
%tRNALysl,2
(spot 2)t
%tRNALys3
(spot 3)6
%tRNALys
%!RNALysl,2
%tRNALys3
2.1
1.0
2.6
5.7
1.2
B. SVC21 BHIO
24.7
5.3
31.2
61.2
1.0
C. SVC21 P(-)
19.6
5.6
25.7
50.9
1.0
1.9
0,4
1.9
4.2
1.2
E. pSVGAG-RRE
2.0
0.6
2.1
4.7
1.2
F. pSVGAGPOLRRE-R P(-)
20.5
5.2
24.4
50.1
1.1
D. SVC21 MSCI
• Determined through phosphor-imaging analysis of the 2-D PAGE patterns in figure 2.
y Spot 1 in figure 2,
t Spot 2 in figure 2.
6 Spot 3 in figure 2.
•
Figure 3. A. Quantitation of tRNALys3
in viral RNA.
The specificity of a
tRNALys3 DNA oligomer probe has been
described previously (15).
Purified
human placenta tRNALys3 is used as an
external standard. Aliquots from the
sarne viral RNA sarnples used to
quantitate genomic RNA in Figure 2 were
used to de termine the arnount of tRNALys3
present. Volumes of viral RNA sarnples
used for hybridization are:
B, 2 ul;
C, 2 ul; D, l ul; E, 2 ul; F, 2 ul.
•
•
A
(ng)
0.1 0.2 0.6 1.0 5.0
tRNALys3
Viral RNA
••
samples
B
C
••
0
E
••
F
125
B
~ 100
-...
>.
:=
~
QI
-
.5
QI
~
75
50
.!li
QI
a:
25
o
1
234
tRNALys3 (ng)
•
101
5
6
Figure 4. A. Quantitative reverse transcription polymerase chain reaction.
Reverse transcription and PCR
is performed on in vitro transcribed genomic RNA (used as
an external standard) or on known volumes of RNA samples
isolated from virus produced by transfecting COS cells with
constructs B-F, as designated in Figure 2. Volumes of
viral RNA samples used are: B, 0.1 ul; C, 0.1 ul; D, 0.1
ul; E. 1 ul; F. 1 ul. Amplified products are run on a 8%
PAGE with 7 M urea to separa te amplified products from
excess radioactive primers.
Equal amounts of viral RNA
samples are also used for PCR amplication without reverse
transcription as a negative control.
B. Genomic RNA RT-PCR standard curve. The relative
intensities of amplified signaIs from in vitro transcribed
RNA are used to plot a standard curve. Concentrations of
genomic RNA from the viral RNA samples are calculateà from
this curve.
•
•
•
ï="
a:
-
IL
:::1
0
W
:!
0
a:
0
::
<1:
Z
..
ii
:>
III
r-
IL
oC
w
<1:
Z
0
e
:>
0
a:
:-!
a:
<1:
Z
a:
'2
III
60~Xl:
60~X~
'"
U'"l
sO~XS
0
sO~X~
'C
-..
C
al
:;
BO~X&
LO~XS
•
1
1
•
1
,
V)
N
a
!il
1
V)
••,
0
V)
U
al
0
:::E
LO~X~
'"
sO~X~
et
•
m
N
" (;OL)'"AqlUllUI
0A1I1I1111
•
•
Table 2. Molecules of tRNA LysJ per 2 molecules of genomic RNA
RNA
. molecules of genomic RNA
source
peT ul sample a
ng of tRNALys3 pcr
ul sampleb
molecules of tRNALys3
peT ul samplec
molecules of tRNALys3
2 molecules of genomic RNA
8
2.0 x 109
0.34
8.0 x 109
8
C
2.3 X 109
0.34
8.0 x 109
8
D
3.0 X 109
0.06
1.4 x 109
1
E
1.7 X 108
0.03
8.5
x 108
10
F
2.0 X 108
0.46
1.1
x 1010
109
a Values Iisted are determined from figure 3.
b Values Iisted are determined from figure 4.
CA tRNA molecular weight of 25000 g/ mole is used to calculate number of tRNA molecules.
•
2.4. Resu1ts
We have previously shown that the tRNALyo isoacceptors, tRNALyol. 2,
and tRNALys3 , are the major abundance tRNAs that are incorporated ioto
infectious virus particles produced from COS-? cells transfected with
HIV-1 proviral DNA
involved
in
the
(16) .
To identify the specifie viral proteins
selective
packaging
of
these
tRNA molecules,
we
transfected COS-? cells with a series of wild type and mutant HIV-1
expression plasrnids.
The construction and characteristics
of
each
expression plasmid are described in the Materials and Methods section,
and the relevant portion of each plasmid is shown in Figure 1.
The
plasmids include three HIV-1 proviral clones which express l)wild type
Prss gag and Pr160gag-pol which are processed (pSVC21 BH10), 2)unprocessed
Prss gag and Pr160gag-pol (pSVC21 P(-), or 3)wild type proccssed Prss gag
products and a severely deleted Pr160g a g-pol which lacks 95% of the RT
domain and approximately 1/3 of integrase(pSVC21 MSC1).
clones
also
express
Pr160gag-pol
aU
of
the
coding region.
viral
proteins
These proviral
downstream
of
the
In addition,
two SV40 la te expression
plasmids that express either unprocessed Prss gag alone(pSVGAG-RRE-R) or
both unprocessed Prss gag and Pr160gag-pol (pSVGAGPOL-RRE-R P (-») were
used.
To
an.,lyze
particles,
the
low
total RNA was
electrophoresis in a
molecular
extracted,
weight
RNAs
end-labeUed,
present
in
viral
and subjected to
tl.O dimensional gel system as described in the
Materials and Methods section. Figure 2 shows the 2D-PAGE patterns of
tRNAs extracted from normal COS-? cells, as well as from virus particles
produced from the cells transfected with the different vectors.
2A shows
the
studies have
tRNA Lyo3
tRNA extracted
from uninfected COS-? ceUs.
identified spots
(16).
1 and 2 as
tRNALyol. 2,
Figure
Previous
and spot 3 as
To quantitate the amount of tRNA present in each spot,
the gel was subjected to phosphor-imaging analysis. Table 1 shows the
quantitation
of
the
tRNALys
spots
from
the
various
gels.
In
untransfecled COS-? cells(Figure 2A), approximately 6% of the cellular
•
tRNA was represented by tRNALys,
close to 1
(Table 1).
and the tRNALYsl,2:tRNALys3 ratio was
In contras t,
in wild type virus (Figure 2B),
approximately 60% of the low molecular weight RNA was tRNALya.
105
Thus,
•
there
W45
a 10 fold increase in the relative concentration of tRNALys in
the virus compared ta the cytoplasrn of COS cells,
selectively incorporated into the virus.
i. e.,
tRNALys was
The tRNALysl.2: tRNALys3 ratio
remains similar to that found in the cytoplasm of COS cells, indicating
that both major tRNALys isoacceptor families are packaged with equal
efficiency.
Because HIV is assembled from the precursor polypeptides Pr55g og
and PrlGOgog-pol, which are cleaved into multiple proteins during or
after viral budding, it was of interest ta de termine if the precursor
Molecules
tRNALys,
themselves
could mediate
the
selective
incorporation
of
or if proteolytic cleavage was nacessary to facilitate the
specific incorporation.
Thus the tRNA pattern was examined in particles
produced from a proviral clone lacking an active viral protease(pSVC2l
P(-».
This clone contains an inactive protease due to a Asp25 to Arg25
change in the active site, and the particles produced trom transfected
cells remain in the immature forro, containing bath unprocessed PrSS gag
and
PrlGOgog-pol
(12).
As
shown
in Figure 2C
and
in Table
l,
approximately 50% of the low molecular weight RNA is tRNALys, with a
tRNALysl,2: tRNALys3 ratio equal to 1.0.
PrlGOgog-pol is the most likely precursor protein to be involved in
tRNALys packaging because it contains the sequences for RT, a protei.n
whose mature farro has been shown ta
(1,32) •
interact with tRNALys3 in
vitro
To investigate the role of PrlGOgog-pol in selective tRNALys
packaging, we transfected COS ceUs with a proviral ONA(pSVC21 MSCl)
which is missing 95% of RT sequences and approximately 1/3 of integrase
sequences (Figure lC). In this mutant, pr55 gog is processed. Figure 20
shows the the low molecular weight RNA pattern in the virus particles
produced, and the phosphor-imaging analysis of the gel is given in Table
1.
The results indicate that while the tRNALys isoacceptors are present
in
the
virus,
they are
not
selectively packaged.
The
relative
concentration of the tRNALys in the virus is similar to that found in
the cytoplasm of non-transfected cells, i.e., 4.2%.
The deletion used
in this experiment was too large to allow mapping of the precise region
•
in PrlGOgog-pol involved in selecting tRNALys for incorporation into the
virus, but this data clearly demonstrates the importance of
in the selection process.
lOG
PrlGOgog-pol
•
On the ether hand.
Pr55 gsg ,
virus.
viral particles containing only unprocessed
(and not Pr160gsg-pol), do not selectively package tRNALys into
These particles were produced by transfecting COS-? cells with
DNA construct E (pSVGAG-RRE-R), displayed in Figure l
(34,35).
The low
molecular weight RNA pattern of these virus is shown in Figure 2E, and
phosphor-irnaging analysis of this pattern (Table 1) indicates that no
selective tRNALys incorporation cceurs, i.e., 4.7% of the low molecular
weight viral RNA is tRNALys.
transfecting
These
COS-? cells
particles
(34,35).
We have also produced virus particles by
with DNA construct F (pSVGAGPOL-RRE-R (P-)).
contain both unprocessed Pr55 gsg and Pr160gsg-pol
The low molecular weight RNA pattern of these virus is shown
in Figure 2F, and phosphor-imaging analysis of this pattern (Table 1)
indicates that selective tRNALys incorporation cceurs, i.e., 50% of the
low molecular weight viral RNA is tRNALys.
This data further supports
the importance of Pr160g sg -pol in tRNALys selection.
I t should also be
noted that construct F produces viral genomic RNA lacking the primer
binding
site(as
,therefore,
does
contruct
E
),
and
that
these
experiments
validate earlier results which indicated that selective
tRNALys incorporation into HIV-l does not require the presence of a
primer binding site on the genomic RNA(lG) .
The results in Figure 2 and Table l showed the effect of mutations
in the viral proteins upon the ability to selectively incorporate
tRNALys into virions, but these experiments did not address the effect
of these mutations upon the absolute amounts of tRNALys incorporated
into the virus particles.
Tc investigate this, we measured the ratio of
tRNALys3/genomic RNA in each of the RNA samples isolated from the wild
type and mutant virus.
These results are shown in Figures 3 and 4, and
Table 2.
We have
previously demonstrated
the ability of
a
DNA probe
complementary to the terminal 3' 18 nucleotides of tRNALys3 to hybridize
specifically with this tRNA (lG).
Total viral RNA from wild type and
mutant virus were blotted cnte Hybond fil ter paper, and the amount of
tRNALys3 present in each sample was determined through hybridization to
•
both thGse samples and to known amounts of purified human placental
tRNALys3
(Figure 3).
The amount of genomic RNA in each sample was
10?
•
determined by quantitative peR, as shown in Figure 4.
quantitated by phosphor-imaging analysis, and
The results were
are listed in Table 2.
We tound that wild type virus contains 8 molecules of tRNALys3/2
copies genomic RNA(Table 2, line B).
Similar amounts of tRNALys3 were
found in the protease-negative mutant virus particles(Table 2, line C).
The virus lacking the RT and part of the integrase showed an 8 fold
reduction in the amount of tRNALys3/2 copies genomic RNA(Table 2, line
D) •
On the other hand,
the tRNALys3/2 copies genomic RNA ratio was
unexpectedly high in virus particles produced from the expression vector
producing only PrSSgag(Table 2, line El, since these particles showed no
specificity of tRNALys incorporation. The tRNALys3/2 copies genomic RNA
ratio was also unexpectedly high in virus particles produced from the
expression vector producing both PrSS gsg and Pr 160g ag-pol (Table 2, line
F) .
These particles did show specifie tRNALys incorporation, but the
tRNALys3/2 copies genomic RNA ratio is approximately 14 times higher
than
that
found
in wild type HIV-l.
These high ratios could be
explained if there was a defect in constructs E and F which resulted in
virus particles that had a 10-fold decrease in their ability to package
genomic RNA. Recent data suggest that these constructs do produce viral
particles defective in the packaging of genomic RNA(H. Carlsdottir, M.
L. Hammarskjold, and D. Rekosh, unpublished data) .
•
108
•
2.5. Discussion
As reported above, approximately 60% of viral low molecular
RNA in HIV-I is tRNALys,
tRNALys3.
tRNA,
(39).
wei9~t
with 30% represented by the primer tRNA.
These results are similar to those reported for the primer
tRNATrp, in AMV, using a very different technique, arninoacylation
Primer tRNATrp represents 32% of free viral tRNA in AMV.
We have
also determined the amount of tRNALys3 and genomic RNA present in HIV-I
total viral RNA, and used this data to calculate the nurnber of tRNALys3
molecules/virion, assuming two copies of genomic RNA/virion.
calculations.
By these
wild type HIV-I con tains approximately 8 molecules of
tRNALys3/v irus.
This nurnber is similar to the value of 6-8 molecules of
primer tRNA/virus reported for RSV (33) and AMV(39).
In this paper, we provide evidence supporting the hypothesis that
the
unprocessed
Prl60000-pol
incorporation into HIV-I.
is
involved in selecting
tRNALys for
First, studies with a protease mutant clearly
demonstrate that proteolysis of viral protein precursors is not required
for tRNALys incorporation. A similar conclusion was reached from studies
using the MuLV (6) and ALV (36) systems.
Second, viral particles containing only unprocessed PrSS oag do nct
selectively
incorporate
tRNALYs,
while virus
containing unprocessed
PrSS ooo and Pr1600 00- po1 do. Thus. although mature NC has been shown to
bind to tRNA Lys3 in vitro(1,27), the NC sequence in PrSS ooo does not
appear, by itself, to play a major role in tRNALys selection.
We cannot
yet rule out the possibility that PrSS ooo and Prl60000-pol might interact
cooperatively to selective tRNALys for incorporation.
hand,
the absence in construct F of
On the other
the genes for viral proteins
downstrearn of the vif gene shows that the protein products of these
genes are not required for selective tRNALys incorporation(Rev protein
is made on a
separate plasmid
in our transfection system,
and its
participation in selective tRNALys incorporation, while unlikely, cannot
be ruled out). The ability of the virus containing unprocessed PrSS ooo
and Pr160000-pol to selectively incorpora te tRNALys in the absence of a
•
PBS on the genomic RNA is also consistent with results from other
retrovirus systems which indicate a lack of involvement of genomic RNA
in this process(20,26).
109
•
Finally,
prevents
removal of RT and integrase sequences fram Pr160g a g-pol
selective
tRNALys
incorporation,
reduction in the amount of tRNALys3
RNA.
The
effect
of
the
and
results
incorporated/2
alteraticn
of
deletion
Alteration
in
in
Pr160gag-pol
the
is
large,
conformation
of
Pr160gag-pol
fold
in:
on
this process.
result
Molecule,
tRNALys
2)
in
1)
an
rapid
ga
inability of the damaged Pr160 g-pol
degredation of the Molecule, or J)
rnolecule ta enter the virus.
and could
the
8
copies of genomic
incorporation indicates the importance of Pr160 ga g-pol
The
in an
more
However, our results are consistent with
the evidence that RT sequences are involved in tRNALys selection.
It is
known that mature RT(p66/pS1) interacts with primer tRNA during reverse
transcription(l,J2),
virus (26)
and it has been shown in both the avian sarcoma
and murine leukemia virus (21)
lacking RT fails
systems that a mutant virus
to selectively incorpora te the correct tRNA.
Finer
mutational analysis will be required to de termine if RT sequences within
the Pr160ga g-pol protein are involved in tRNALys selection.
Wild
type
tRNALys3/ v irion.
incorporate
HIV-1
con tains
approximately
8
Molecules
of
Virus es unable to process precursor proteins still
8 Molecules
tRNALys3/ v irion,
while
those lacking RT and
integrase reduce the incorporation of tRNALys3 approximately 8 fold.
Our calculations assume that these mutants incorporate normal amounts of
viral genomic RNA.
This assumption is supported by the findings that
normal amounts of viral genomic RNA are incorporated in a proteasenegative
ALV
mutant(J6)
and
in
RT-negative
avian
and
murine
retrovirus(21,26).
Our resul ts are also consistent wi th our unpublished data
(H.
Carlsdottir, M. L. Hammarskjold, and D. Rekosh) that shows that RNA from
constructs resembling E and F cannot be packaged into particles.
explains
the unexpectedly high ratio of tRNALys3 molecules/2
genomic RNA observed in
plasmid
expressing
incorporation,
genomic RNA.
•
unprocessed
yet
these cases.
only PrSS gag did
contained
11
Particles produced
not
Molecules
show
of
copies
from the
selective
tRNALys3/2
This
tRNALys
copies of
Particles produced from the plasmid expressing both
PrSS gag and Pr160g a g-pol showed selective packaging of
tRNALYs, but contained 109 Molecules tRNALys3/2 copies of genomic RNA, a
ratio
14 fold higher than found in wild type HIV-l,
110
The 10
fold
•
differences
between
in
these
difference
the
two
in
ratio
of
tRNALys3/2
viral
populations
copies of
probably
genomic RNA seen
reflects
tRNALys selective packaging because of
Pr160g s g-po1 in one and not the other.
a
10
fold
the presence of
While it is not clear why the
genomic RNA fram these constructs fails te be packaged,
these results
are consistent with recent findings which indicate that the two ragions
missing in our constructs, the 5' part of the genome through the PBS and
the region 3'
of the vif gene
(34,35),
are required for genomic RNA
packaging (30; E. Vicenzi and M. Martin, personal communication).
The ratio of
cytoplasm
and
in
tRNALysl. 2 / tRNALys3
is similar both in the cell' s
the
indicates
virus.
This
that
these
tRNALys
isoacceptors are packaged with equal efficiency, and suggests that sorne
common feature of
these molecules is recognized by a viral protein
during packaging.
Mature RT has been shown to interact in vitrj with
tRNALys3 (1,2,32,42). Yet this region is quite
different between tRNALys3 and tRNAL y sl,2, differing by 6/17 bases.
If
the anticodon arm of
this
region
packaging,
of
the
molecule
serves
as
a
recognition
signal
for
then the two different sequences in this arm must fold in a
similar conformation.
On the other hand, i t is possible that the
interaction of Pr160 gs g-pol with tRNALys3 in vivo is quite different than
the
interaction
studied
in vitro with mature,
processed RT.
This
difference could be due to artifacts created in the in vitro systems in
which the interaction between mature RT and tRNALys3 are studied, but it
is possible that the interactions involved in packaging tRNALys3 are
quite different
than
reverse transcription.
the interaction between RT and tRNALys3 during
For example,
it has been postulated that the
tRNA binding site on RT may involve both p66 and p51 polypeptides in the
RT dimer (19), but it is not known if Pr160gsg-pol can also form a dimer,
and,
that
if so,
interact with tRNALys in the dimer forro.
tRNALys3
is
interacting
with
packaging and reverse transcription,
two
different
Besides the fact
molecules
during
the tRNALys3 may be in different
states in the cytoplasm and in the virus.
For example, most cytoplasmic
tRNALys3 is expected to be complexed with proteins such as lysine tRNALys
•
synthetase or
the
eukaryotic
elongation
carries tRNA to the ribosome (15,23) .
Hl
factor,
EF-l alpha,
which
It may thereEore be that the
•
Pr160gag-pol binding site on tRNALys3
i5 composed of prote!n as weIl as
RNA.
In retrovirus, primer tRNA is presant in a higher abundance in the
virus than mast ether tRNAs.
ls this higher abundance a requirement for
its efficient function as a primer tRNA, or is efficiency of function as
a primer more influenced by specifie structural properties of the tRNA?
A mutant murine leukemia virus was isolated by Colicelli and Goff[S]
which contained a PBS complementary to tRNAG1n rather than tRNAPro, the
normal
primer
tRNA in
this virus.
This virus was
presumably could use tRNAGln as the primer tRNA.
aIse
obtained
in
which
a
recombinant
murine
retroviral vector containing a PBS for tRNAGlnl
infectious,
and
Similar results were
leukemia
virus-based
or tRNALys3
instead of
tRNAPro also appeared to be replicated using tRNAGlnl or tRNALys3 as a
primer for RT(22).
The concentration of tRNALys relative to other viral
tRNAs is high in many retroviral systems examined, including MuLV(40),
but the relative abundance of tRNAGln in MuLV has not been reported, and
it would be of interest to know i f the selective incorporation of
tRNAGln into the mutant virus or in the retroviral vector particles is
required
for
transcription.
it
to
act
efficiently
As mentioned above,
as
evidence
a
primer
for
reverse
is presented here and
elsewhere that viral genomic RNA does not appear to be involved in the
selective incorporation of primer tRNA, including the MuLV system (20) .
•
112
•
2.6. Acknowledgements
This work was supported in part by grants from the National Health
Research
Development
Program.
Research Council(Canada).
Health
and
Welfare
Canada.
Medical
and by grants HAI30399 and AI25721 from the
national Institutes cf Health. USA.
Thanks
ta Sandy Fraiberg for assistance
rnanuscript .
•
113
in preparation of
the
•
~.7.
1.
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OUesberg,
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•
10.
Paras, A.J. and N.A. Dibb1a. 1975. RNA-directed DNA synthesis by
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functional
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Sei. U.S.A.
11.
72: 859-863.
Paras, A.J., A.C. Garapin, W.B. Lavinson, J.M. Bishop, and
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1973.
Characterization
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H.M.
10w-mo1ecu1ar-weight
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Gottlingar, H.B., J.G. Sodroski, and W.A. Hasaltina. 1989. Ro1e of
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in morphogenesis and
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Proc.
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14.
Harada, P., R.C. Sawyar, and J.B. Dahlbarg. 1975. A primer RNA
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Harshay, John W.B., ed. Translational Control in Mammalian Cells.
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1991, Annua1 Review
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Jiang, M., J. Mak, A. Ladha, B. Cohan, M. K1ain, B. Rovinski, and
L. Klaiman. 1993.
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17.
60.
Identification of tRNAs incorporated into wild type
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Jiang, M., J. Mak, M.A Wainbarg, M.A. Parniak, B. Cohan, and L.
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Biophysics Research Communications.
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18.
Jiang,
M.,
M.A.
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Isolation and
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19.
Kohi.taedt.
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L.A.,
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Crystal structure at 3.5 angstrom reso1ution of HIV-1
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Levin. J.G. and J.G. seidman. 1979. Selective packaging of host
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21.
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22.
Lund. A.H •• M. Duch. J. Lovmand. P. Jorgen.en. and F.S. Pederson.
1993.
Mutated primer binding sites interacting with different tRNAs
allow efficient Murine Leukemia Virus replication.
J. Virol. 67: 7125-
7130.
23.
William C., Mechanism
Merrick.
Protein
Synthesis.
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Petara. G.G. and and J. Hu.
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27.
Prats,
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U.
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Ratner, L., W. HasAleine, R. Patarca,
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Richardson, J.H., L.A. Child, and A.M.L. Lever. 1993. Packaging of
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Rosenthal,
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Amino
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Sarih-Cottin, L, B. Bordier, K. Musier-Porsyth, M. Andreola, P.J.
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palyacrylamide
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gel
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34.
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1993.
smith, A.J., N. Srivivasakumar, M. Hammarskjold, and D. Rekosh.
Requirements
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Stewart, L., G. Schatz, and V.M. Vogt. 1990. Properties of avian
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Taylor,
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1977.
An analysis of the role of tRNA species as
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Waters, L.C. 1978. Lysine tRNA is the predominant tRNA in murine
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Biochim.
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Biochem. Biophys. Res. Commun. 81: 822-827.
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1977.
Transfer RNA in RNA tumor
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Waters, L.C., B.C. Mullin, T.Ho, and W.K. Yang. 1975. Ability of
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Witte,
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1978.
Relationship
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J. Virol. 26: 750-761.
Wohrl, B. M., B. Bhresmann, G. Keith, and S.F.J. Le Grice. 1993.
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•
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transcriptase/tRNALys3 Complexes. J. Biol. Chem. 268: 13617-13624 .
'
118
tryptop
•
Preface(Chapter 3)
In the previous chapter, we
h~ve
shown that the Gag-Pol precursor
protein is involved in the selective packaging of primer tRNALys3 into
HIV-I viral particles, and provided evidence which supports a role for
RT sequences in this process.
In this chapter, mutational mapping was
done on different regions of Gag-Pol{RT and IN) to determine regions of
the Gag-Pol precursor which are involved in the selective incorporation
of viral tRNALys.
We find that only mutations in the connection domain
or in the region immediately N-terminal of the connection domain of RT
inhibit selective tRNA incorporation.
We also find a direct correlation
between the 'bsence of selective tRNA packaging and the absence of
precursor protein processing in the virus .
•
119
•
Chapter 3
RT Connection Domain Mutations in pr160gag-pol znhibit the
Selective Zncorporation of Primer tRNALY83 into arv-l
•
120
•
3.1. Ab.tract
During HIV-1 viral assemb1y, both Pr160g ag-pol and primer tRNALys3
are packaged into the virion.
Select tRNALys packaging(both tRNALys3 and
tRNALysl. 2) is dependent upon the presence of RT sequences wi thin
Pr160 ga g-pol. In this work we have monitored the effect of Pr160gag-pol
mutations upon selective tRNALys3 viral incorporation, using Cos-7 cells
transfected with mutant HIV-1 provira1 DNAs as the source of mutant
virus. Mutations include carboxy terminal deletions of Pr160gag-pol and
sma!1
amine
acid
insertions
functional domains of the RT.
and
replacements
within
the
various
tRNALys3 incorporation was monitored both
by 2D PAGE of viral RNA, and by hybridization with tRNALys3- spec ific and
genomic RNA-specifie DNA probes.
Our data indicates : 1) Deletion of
integrase sequence has only a small effect upon selective tRNALys3
packaging, while carboxy terminal deletions extending further into the
RNAse H and connection domains prevent selective tRNALys3 incorporation;
2) Select tRNALys3 packaging is inhibited by small in-frame amino acid
insertions or replacements within or flanking the RT connection domain.
but tRNALys3 incorporation is altered little or not at all by similar
amine acid insertional mutations within the fingers,
RNase H domains of RT.
palm,
thumb,
or
The viral content of mature Gag and Pol pro teins
was also examined in the mutant virus,
using western blot analysis.
A
direct correlation was found between the ability of mutant virus to
selectively incorporate tRNA Lys3 and the content of mature viral protein
species, which may indicate an inability of Pr160 ga g-pol mutated in the
connection domain to be incorporated into the virus or of the viral
protease to carry out proteolytic processing of viral precursors .
•
121
•
3.2. Introduction
A subset of host cell derived tRNAs are packaged into retrovirus
during viral assembly(9,
10,
18,
31,
42,
45).
The 3 '-terminal 18
nucleotides of one of the packaged tRNAs is complementary to a region
near the 5' end of the 35S RNA genome which is termed the primer binding
site(PBS). The bound tRNA is known as the primer tRNA because it is used
to
prime
the
retroviral
reverse
minus-strand
different retroviruses,
thus
far
transcriptase
been
isoacceptors,
DNA.
(RT)-catalyzed
Different primer
synthesis
tRNAs
are
of
used by
and the natural primer tRNAs discovered have
confined
to
tRNAPro
tRNATrp,
and
tRNALysl,2 and tRNALys3 (for review,
see
the
tRNALys
(25».
The PBS
sequence in human immunodeficiency virus type 1(HIV-1) suggests that the
primer tRNA in this virus is also tRNALys3(37) , one of the three major
tRNALys
isoacceptors
in mammalian cells (36).
During viral ass"mbly,
tRNALys isoacceptors are selectively incorporated into the HIV-1 viral
particles
in
(18, 27).
Pr55 gag and Pr160gag-pol are two m~jor precursor proteins involved
the inital assembly of HIV-l. During viral budding, Pr55 gag and
Pr160 gag -pol are cleaved to mature proteins by a viral protease present
in Pr160gag-pol (22)
Previous reports have shown that expression of
gag
l'r55
precursor protein provides sufficient information for
virus
particle formation and release from the host cell membrane (48) .
The
viral
incorporation of Pr160gag-pol appears to be dependent upon the
myristylation of the Pr55 gag precursor protein, and independent of
Pr160g a g-pol
incorporation
that
Pr160g a g-pol
is mediated through myristylated Pr55g a g.
Pr160g a g-pol
myristylation(30,
46). This
suggests
has been shown to be involved in tRNALys3 packaging into the virus, and
selective tRNALys incorporation occurs independently of RNA packaging or
precursor processing(27).
RT sequences within the precursor molecu1e
represent a likely site for tRNALys3-Pr160gag-pol interaction, because 1)
mature RT has been shown to interact with tRNALys3 in vitro(3, 44), and
2)
•
the
absence
of
RT
has
been
shown
to
al ter
the
selective
incorporation of primer tRNA in avian(32), and human(27) retroviruses.
In this work,
either
we have examined a series of mutants which are
carboxy terminal deletions of Pr160gag-pol, or small ami no acid
122
•
insertions and replacements within the
RT sequences of Pr160g ag-pol. The
mutations are placed within the context of HIV-l proviral DNA, and these
DNAs are
transfected into Cos-7 cells.
The tRNALys3 content and mature
viral protein patterns in the resulting mutant viral particles were
analyzed.
Our data
flanking
the
indicates
connection
that mutations specifically within or
domain
of
RT
prevent
selective
tRNALys3
packaging, and reduce the viral content of mature Gag and Pol protein
sequences .
•
123
•
3.3. Materi&l. and Method•
Plaamid. Con.truction
All plasmids used in this study are derived from SVC21 BH10(27).
Transfections of these plasmids into COS-7 cells yield wild type viral
proteins and with various mutated Pr160 gag -pol precursor protein.
Schematic representations of various mutated Pr160gag-pol used in this
study are illustrated in Fig. 1.
A 6 nuc1eotide linker(5'TGGCCA3') containing an MSCI restriction
site
was
cloned
into
the
SmaI
site
of
PSVK3(Pharmacia), and it was renamed as PSVK3 MSCI.
expression
plasmid
A fragment of HIV-1
Sequence(2615-4550) was removed from SVC21 BH10 with the restriction
enzyme MSC1(New Eng. Biolab).
This HIV-1 fragment con tains 95% of the
RT sequence and 1/3 of the IN sequence, and it was cloned into the MSCI
site of the PSVK3 plasmid.
The PSVK3 derived plasmid containing the
HIV-1 RT sequence is renamed as PSVK3 RT.
Multiple cut enzymes DraI(Pharmacia) and RsaI(Pharmacia) were used
to randomly digest PSVK3 RT plasmid.
Slices of agarose containing
linearized plasmids with a single and double cut were cut out from 1%
agarose gel.
DNA fragments were electroeluted from agarose and were
purified with the Wizard DNA Clean Up System(Pt"omega).
Purified DNA
fragments were then ligated in the presence or absence of a 6 nucleotide
phosphorylated linker NaeI (5 'GCCGGC3') .
Ligation products were then
transformed to bacteria TGl, and plated in the presence of ampicillin.
Colonies were selected and
amplified.
Mutant plasmids were detected by
the alternation of DraI or RsaI digestion patterns, compared with those
of the wild type PSVK3RT.
Plasmids with mutations found in the HIV-l RT
sequence were selected, amplified, and the corresponding HIV sequences
were cut out with MSCI and recloned
back to SVC21 MSC1(27).
Mutants SVC21 DrdI, SVC21 DrdII, SVC21 Dr2, SVC21 RdI, and 3VC21
R3b were constructed in the above manner.
DNA sequencings and enzyme
digestions were done to confirm the mutations and the orientation of the
inserts.
•
The out of frame deletions of SVC21 DrdI (129bp deletion),
SVC21 DrdII (947 bp deletion), and SVC21 RdI (397 bp deletion) crellted
non-HIV coding sequence beyond the 5' deletion point, which resulted in
new termination codons 35, 16, and 1 amine acid, respectively, after the
124
•
deletion start sites.
Additionally, SVC2l DrdI and SVC2l DrdII resulted
from failed attempts to create insertion mutation, and the initial nonHIV coding sequence immediately after the deletion start point con tains
the 5 and 6 bp inserts, respectively.
SVC2l A2a, SVC2l A4, SVC2l Ha4, and SVC2l Ra were constructed by
recloning
the
corresponding
fragments from full
2kbp
MSCI(HIV-l
Sequence,
length HIV-l plasmids A2a/XA,
Ra/XA to SVC2l MSCI.
A4/XA,
2615-4550)
Ha4/XA, and
DNA sequencings and enzyme digestions were do ne to
confirm the mutations and the orientation of the inoerts.
The integrase deletion mutant,
SVC21 BspMI,
was constructed by
removing DNA sequence within the BspMI sites in SVC2l BHI0.
was
first
digested with
BspMI,
and
the
746bp
BspMI
SVC2l BHlO
fragment
was
separated from the remaining l4kbp fragment in a 1% agarose gel.
l4kbp
fragment
was
electroeluted and purified.
The
An oUgo adaptor
containing a new translational termination site(5'TGTATAGGGTCA3') was
mixed with
adaptor
the
we
l4kbp fragment
first
mixed
for
Ugation.
equal
To prepare the oUgo
molar
amounts
of
BspMI
A1 (5'TGTATAGGGTCA3'), and BspMI A2(5'TGTTTGACCCTA3') in 25mM Tris-Cl, pH
a.o
and
10mM MgC12.
minutes,
and
it
Ugating
the
oligo
The oUgo mix was
then heated at 95°C for
was slowly cooled down
adapter
to
the
14
to room temperature.
kb
fragment,
5
After
bacteria were
transformed with the ligation mix and the plasmids were amplified in the
bacteria.
The plasmid DNAs were sequenced to confirm the presence of
the deletion and the new termination codon.
SVC2l BspMI expresses a
truncated Pr160ga g-pol sequence up to amine acid 30 of the IN sequence,
which is immediately followed by a termination codon, UAG.
The remaining five mutants were constructed using recombinant
PCR(15) .
Heat stable polymerase Pfu(Stratagene) was used for 'Ill PCR
reactions in this study, and DNA sequencings were done on 'Ill amplified
DNA fragments to confirm the presence of mutations and fidelity of the
enzyme.
No spontaneous mutation was found in any of these PCR products.
Recombinant
products (FI
•
PCR
,
F2)
requires
amplification
which overlap in sequence.
joining and extension of
reaction.
independent
the FI and F2
This is
fragments
of
two
PCR
followed by
through the PCR
A subsequent reamplification of this fragment with only the
125
•
right- and left-most primers results
in the enrichment of the fu11-
length, secondary products.
Eight of these mutant RT sequences (R3, R6, A2a, Ha4,
H2,
R7,
R8
and A4) were initally constructed and expressed in the RT expression
vector,
PHRTRX2(35).
recloned
into
Four of these mutants(R3,
SVC21
MSCI
as
follows:
RT5' Os (5' CCCATTAGCCCTATTGAGAC3' ) ,
(5' CCTGATTCCAGCACTGAC3 ')
tem;:>lates,
and
sites
were
R6,
H2
sense
primer
and
R7 (35)
primer
RT3 'Pa
as
DNA
HIV-l RT sequences containing the
amplified.
Using
RT3' Ps (5' GTCAGTGCTGGAATCAGG3'),
RT(Del)a(5'CCCGCCCACCAACAGGCG3'),
H2, and R7) were
Using
anti-sense
PHRTRX2: R3 ,
fragments of 1673bp(Fl)
mutational
R6,
sense
antisense
primer
primer
and SVC2l BHIO as DNA templates,
fragment of 430bp(F2) was amplified.
a
The desired FI and F2 fragments
were then mixed, joined, extended, and reamplified.
The final secondary
PCR
and
products
were
phenol-chloroform
restriction enzyme MSCI.
extracted
digested
with
The digested secondary products were separated
by running on a 1% agarose gel, and the 2kbp MSCI fragment was purified
and subcloned back to SVC2l MSCI.
SVC21 GR was also cons tructed through recombinan t
amplified
by
using
sense
primer
RT5'OS,
PCR.
FI was
antisense
primer
RT3'IA(5'TGTTTCCTTTTGTATGGGCAGTTT3'), and SVC2l BHlO as DNA template.
F2 was amplified by using antisense primer RT(Del)A, SVC2l BHlO as DNA
template, and a 99 nucleotides long oligo sense primer corresponded to
HIV-l DNA sequence 3716 to 3814 with the alternation of nucleotides
3740-2,
3749-51,
3752-4,
3764-6,
TGG(Trp) to GAG (Glu) ( see Fig. 1).
reamplified as
3776-8,
and 3788-90,
which
change
FI and F2 were joined, extended, and
previously described(15).
The
final
secondary PCR
products were phenol-chcloform extracted and digested with restriction
enzyme MSCI.
The digested secondary products were separated by 1%
agarose electrophoresis,
and the 2kbp MSCI fragment was purified and
subcloned back to SVC2l MSCI.
Production of wild-type and mutant Hrv-l
COS-7 cells were transfected with wild-type and mutant proviral
•
DNAs by the calcium phosphate method, and virus was isolated from the
culture medium 63 h post-transfection as previously described(27) .
126
•
RNA analysis
Isolation of viral RNA, purification of human placental tRNALys
isoacceptors,
RNA labeling, one and two dimensional polyacrylamide gel
electrophoresis (ID and 2D PAGE), and measurement of tRNALys3 by RNA-DNA
hybridization
~ere
performed as previously described(271 .
Tc quantitate the amount of genomic RNA in viral RNA samples. an
antisense oligo 790H(5'CTGACGCTCTCGCACCC3') was used.
790H hybridized
specifically to HIV-l genomic RNA sequence 338-354 (DNA sequence 791807) .
Dot
blot
hybridizations
were
performed
using
Hybond
N
paper(Amersham) with either in vitro-transcribed HIV genomic RNA as a
standard or total RNA from wild type and mutant viral particles.
The
DNA oligomer was first S'-end labeled using T4 polynucleotide kinase and
gamma- 32 p-ATP (3000 Ci/mMol, Dupont Canada), and specifie activities 10 8
to 10 9 cpm/ug were generally reached.
Approximately 10 7 cpm oligomer
was generally used per blot in hybridization reactions.
A standard
curve is established by using known amounts of in vitro-transcribed RNA
for hybridization reaction.
A 957 base RNA fragment
(sense)
from a linearized DNA plasmid, pEA2, with T7 RNA polymerase.
fragment corresponds to HIV-l 3B DNA sequence 473-1420.
genomic RNA in wild type,
is made
This RNA
Quantitation of
mutant viral RNA samples,
and
transcript RNA are done by phosphor-imaging (Bio-Rad).
in vitro
The relative
intensity of amplified signaIs is used to determine the concentration of
genomic RNA in the viral samples using the standard curve.
Characterization of wild-type and mutant viral particles
63 h post transfection, the cell culture supernatant was clarified
by centrifuging in a Beckman Gs-6R rotor at 3,000 rpm for 30 min at 4°C.
Aliquots
of
clarified
viral
transcriptase activity Assay,
supernatant
were
saved
for
p24 antigen captured Assay,
reverse
and viral
infectivity assay.
(il Reverse transcriptase activity assay
50ul of clarifed supernatant was mixed with 50ul RT mix containing
50mM Tris-Cl, pH8.0, 5mM MgC12, 150mM KCl, 0.5mM EGTA, 0.05\ Triton X100, 2\ ethylene glycol, 5mM dithiothreitol. 0.3mM reduced glutathione.
•
50ug/ml poly(rA)ooligo(dT).
80Ci/mmol).
and 20uCi
[3HldTTP(spec.
activity 50 to
The reactions were incubated at 30°C for 22 h.
l ml of
cold 10\ TCA(in 20mM sodium pyrophosphate) was added to the reaction
127
•
mixed
ta
terminate
the reaction,
and precipitated for
2 h at 4°C .
Incorporated [3HldTTP was precipitated cnte Whatman GF/C filters, which
were then counted for radioactivity in a Packard tri-Carb scintillation
analyzer.
(ii) p24 antigen captured assay
Clarified supernatant was diluted and assayed using p24 ELISA
detection kit(Abbott, Diagnostics division).
Assay conditions were clone
according ta the manuafacturerls recommandation.
(iii) Tissue culture infective dosage-SO%(TCIDSO) assay
SOul of clarified supernatant was used for each infectivity assay
to
infect MT4
target
cells.
Each viral
sample was
tested at
10
different dilutions,each dilution being replicated eight times.
The
virus-induced cytopathic effect(CPE) was used to score
The
reaction
conditions
described(19) .
of
The assay
the
TCIDso
assay
were
postives.
as
previously
allows for the detection of the wild type
HIV-l virion, SVC21 BHlO, at concentrations
as low as 0.2ng
01
p24/ml
of clarified supernatant.
Western Analysis
Transfected cells were washed twice with phosphate buffer saline,
and
cellular prote in was
extracted with
IX
RIPA buffer.
Sucrose
cushion-purified viral particles were washed with IX TNE and viral
protein was extracted with IX RIPA buffer.
viral
prote!n,
described(S).
and
Western
analysis
Extraction of cellular and
were
clone
as
previously
AlOS patients sera and 12SI-protein A/G(ICN) were used to
detect viral proteins .
•
128
•
Figure 1. Schematic representations of
wi1d type and mutant Pr1Gogag-pol precursor
proteins. DrdI, DrdII, and RdI are
de1etion mutants that express truncated
Gag-Pol precursor pro teins up to the
connection domain of the RT.
BspMI is an
integrase deletion mutant that expresses
truncated Gag-Pol precursor up to the
first 30 ami no acids in the integrase
sequence. R3a, R3b, RG, A2a, H2, R7, Ha4,
Dr2, Ra, and A4 are in frame linker
insertion mutants, and the sizes of
nucleotide inserts are indicated in the
parenthesis. GR is a replacement mutant.
A stretch of trytophan residues is
replaced with glutamic acids as indicated
in the figure.
* represents the mutations
that affect selective tRNALys3
incorporation, and viral content of
processed Gag and Pol proteins.
•
•
Pr160 gag-pol
MA
CA
Ne
RT
PR
IN
:;:;:;:;:;:::
lingers palm fingers
I)r
palm
thumb
..
'2'.yT...y.:!'2'<!z!>yT."'yT.d....
connectlon
IN
--'
RNaseH
dl"
Dr dU"
R dl"
8spMI
~;:;:;:;:;:;:;~T~Tgrg:?'C
1
fingers palm fingers
palm
thumb
ccnnectlon
.....J
RNaseH
T(6)
R3a
T(6)
R3b
-..TyTiP;,TZ!lL
~~.:.:.:::.:.::~'Y'T.'T;:r.'''''
_ _.
. . J ~ " " " •••••
_-----J __
fingers paim Rngers
palm
thumb
connectlon
l
----J
RNaseH
_
GlnLysGluThrTrp GluThrTrpTrp ThrGluTyrTrp GlnAlaThrTrp lIeProGluTrp GluPheValAsn
•
GR"
GlnLysGluThrGlu G1uThrGluGlu ThrGluTyrGlu GlnAlaThrGlu lIeProGluGlu GluPheValAsn
•
Table 1. Mutations in HIV·1 Pr160g a9-pol
Amino acjd changes
Mutants a
•
Location b
Mutations(bp}b
From
Ta
B, SVC21 Drdl
358S
D(i29},1(5}
C, SVC21 Drdll
358S
D(947}, I(u,
D, SVC21 Rdl
3827
D(397)
E, SVC21 BspMI
4317
D(724), 1(8)
F, SVC21 R3a
2752
I(S)
S
SEF
G, SVC21 R3b
2752
I(S}
S
SAG
H, SVC21 RS
3270
I(S)
V
VNS
l, SVC21 A2a
3300
I(S)
S
RNS
J, SVC21 H2
3472
1(9}
a
alEF
K, SVC21 R7
3489
I(S)
V
VNS
L, SVC21 Ha4
3552
1(12)
G
GANSR
M, SVC21 Dr2
3715
I(S)
F
FAG
N, SVC21 R8
3827
D(1},1(7}
Y
SRF
0, SVC21 A4
3884
I(S)
A
AGIP
P, SVC21 GR
3740
R(18)
W
E
a Mutant names are preceded by letter designations identical to those used to label panels in Figure 2,
b The nucleotide location is based on the HXB2 HIV·l proviraJ DNA sequence. D, deletion;
l, insertion; A, replacement. The first four mutations in the table(B,C,D, E) contain 3' deletions
of Pr160gag-pol coding .sequence. The position of the 5' terminus of each deletion is IIsted
under 'Location'. In the 'mutations' column, the length of the deletion is listed in the flrst parenthesis,
and any added inserted bases are Iisted in the second parenthesis. The last mutation in the table, P.
designates a cluster of 6 tryptophan amino acids(beginning at 3740}, separated on average by 3
amino acids, which have been replaced with 6 glutamic acids(a tolal of 18 bases replacement). The
remaining mutations, except for N, represent 2·4 amino acid insertions at the location designaled.
N was constructed by deleting one base and inserting 7 bases at the location Iisted.
Figure 2. 2-D PAGE patterns of low molecular weight viral
RNA. Electrophoretic conditions were as described in the
text.
(A) wild type virus(SVC21 BHI0); B to P, mutant
viruses: (B),SVC21 DrdI ; (C),SVC21 DrdII ; (D),SVC21 RdI
; (E),SVC21 BspMI ; (F),SVC21 R3a ; (G),SVC21 R3b;
(H),SVC21 R6 ; (I),SVC21 A2a ; (J),SVC21 H2 ; (K),SVC21 R7
; (L),SVC21 Ha4 ; (M),SVC21 Dr2 ; (N),SVC21 RB ; (O),SVC21
A4 ; (P),SVC21 GR .
•
•
•
•
....
.. ~
Q
":'.,
t'", -
.'",."
'. :i,:.'."..
H:<
'!l'''f
•
ft
ft
•
.
"",
.
•
:'" co
.:~
·.,{,:'~~~,lfij~·;)~~~&~\~7t~*:
. :•. ',é~
:""':''iî'l0JY.6X~\'';~'''~;'·i'''~{W;;~~
":J~~i:;~~@?~tstj:'~:11~;~lg!f;;~tl~
,
"
;~
..
,
...:2:
•
•
Table 2. Characterization of Wild·Type and Mutant HIV·~ Viral Particles.
Samples
RT activity(cpm)a p24(ng/ml)b TCID 50 liter/mie
16374.0
0.0
N.1.
NA
NA
284710.5
68.4
32768.0
9581.29
3923
B, SVC21 Drdl
18821.0
43.7
N.1.
NA
56
C, SVC21 Drdll
15319.5
19.6
N.1.
NA
NA
D, SVC21 Rdl
13589.0
56.3
N.1.
NA
NA
E, SVC21 BspMI
526169.0
136.1
N.1.
NA
3746
F, SVC21 R3a
454038.5
202.9
6888.6
679.02
2157
G, SVC21 R3b
629785.0
249.8
19484.0
1559.97
2456
H, SVC21 R6
210915.5
298.3
181.0
12.14
652
l, SVC21 A2a
164713.5
65.2
13777.2
111.05
2275
J, SVC21 H2
14865.5
19.8
N.1.
NA
NA
K, SVC21 R7
21838.5
28.3
N.1.
NA
193
L, SVC21 Ha4
12459.0
2.8
N.1.
NA
NA
M, SVC21 Dr2
15709.5
60.3
N.1.
NA
NA
N,
SVC21 R8
14952.0
111.3
N.1.
N.I\.
NA
O,SVC21 A4
52784.5
194.2
N.1.
NA
lB8
P, SVC21 GR
15272.0
38.7
N.1.
NA
NA
Background
A, SVC21 BH10
•
Relative aclivily per ng of p24 d
TCID liter
RT aclivily
50
a50ul 01 cell-Iree supernatant was used to analyze the RT activity lor each viral sample.
bl00 to 1000 lold dilution 01 cell-Iree supernatant were used for quantitation 01 p24 value.
e50ul 01 cell-Iree supernatant was used lor TCIDso analysis lor each viral sampie, and
the value is scored at day 6 post-inlection. The given condition allows lor the detetction
01 as low as O.2ng 01 p24/ml 01 cell-Iree supernatant containing wild type(SVC21 BH10)
viral particles. N.I.(Non-lnlectious) N.A.(Not Applicable, either because of virus is not
inlectious or RT activity is background).
dRelative activities were measured by subtracting the background Irom the measured
activities, and the results were normalized to the p24 value..
•
Table 3. Effects of Mutations upon 2·D PAGE Viral tRNA Patterns and
tRNALys3 Incorporation
-.
Viral ANA
A, SVC21 BH10
2-D PAGE Pattern a
tANALys3/genomic ANA:
% of wild typeb
++
100
B. SVC21 Drdl
16
C. SVC21 DrdlJ
14
D. SVC21 Adj
14
+
64
F. SVC21 A3a
++
109
G, SVC21 A3b
++
104
H, SVC21 A6
++
110
l, SVC21 A2a
+
64
E, SVC21 BspMI
J, SVC21 H2
K. SVC21 A7
18
+
99
L, SVC21 Ha4
19
M, SVC21 Dr2
17
N, SVC21 A8
+
66
0, SVC21 A4
++
103
P, SVC21 GA
21
aThe 2-D PAGE patterns of viral ANA have been divided into three categories:
++, tANALys pattern is clearly visible;
+, tANALys pattern is detected with increased background;
•
b The
-. tANALys pattern is difficultto detect because of high background:
tANALys3/genomic RNA ratio are determined from figures 3 & 4.
Figure 3. Quantitation of genomic RNA in viral RNA
samples. A. Dot blots of either known volumes of viral RNA
samples or known amount of in vitro transcribed genomic
RNA are used for hybridization. The blots are hybridized
with a DNA oligomer(790H) complementary to HIV-1 genomic
RNA sequence 338-354(DNA sequence 791-807) . B. The
relative intensities of hybridization signaIs of in vitro
transcribed RNA were used to plot a standard curve.
Concentration of genomic RNA from tne viral RNA samples
were ca1cu1ated from this curve.
•
•
•
<
~=
~
~
s~
"a
u
u
"0
" e
-<:
~
0
<
~
~
~
0
"
(\111) ,(1!SU;'jIUIô'l"!lCI:tll
ID
~o!>
.s>~
"e':
là'>~
v'l
"'<!) v'l 0$~~" oS'.
OJ,ct:Jo!>
Q) "oS'
"ct:J ..Y
"e': oS'.
"oS'
~
{()~
1<'0", "ct:J'1.
0"
(()
'0
0"
~
••
••
"ct:J oS'1"oS'
1-
•
••
•
"'o!>
oS'.
0
"oS'
"~ b
"ct:J <9
"oS'
b-
•
*
•
•
<
z
a:
~
°'0
....+
O~O
sa
....+
...,....+"
...,oS'
....+
...,....+"
...,oS'
'S-"
0$-19 "oS'
•
"e':
"'b- v'l
"e': oS'.
09~ v'l 0"e': oS'.
~O v'l 0
<~ ct:J'l
"e': oS'.
<%- v'l )
" oS'.
Il> "v +
~ "oS'
"e':
v'l
;..
oS';.
....+
'.(
<0....+"
•
<0 oS'
••
«
z
a:
~
t
••
"
....+
.s>o"
....+
"
<
-z
0a:
.
';"2
"
lil"
ëiJjj
::;l/l
Figure 4. Quantitation of tRNALys3 in viral RNA samples. A.
Dot blots of either known volumes of viral RNA samples or
known amount of purified human placental tRNALys3 are used for
hybridization. The blots are hybridized with a DNA oligorner
cornplernentary to the 3' terminal 18 nucleotides of tRNALys3.
B. The relative intensities of hybridization signaIs of
purified tRNALys3 were used to plot a standard curve.
Concentrations of tRNALys3 in the viral RNA samples were
calculated frorn this curve.
•
•
•
~
"
0;,
2
.s
;:l
'"9c
]
~
0
:0
m
..
=:
:!
c
(çO 1) Âl!SU:tlUI ôtA!l UI;J:lI
096-
JO.
~
Z
<;!;
c
c
6-é)
là>6-"'~-t
...~ iS'.
\ . 't .y
,
...~O'~
&... '10'.
.." ..,
'10'
~
••
••
"/~-t
..."" 0' •
096- "-t ...
..."" 0'.
~Q "-t 0
..."" 0'.
""1iS'~
'"
.
06-
~
~4 ..." ,
0,. '10'.
~~Q
o o...~'10' .
"
~& '10'
...~ ~
'10'
;:
1:
~
~
•.
•
•
•
••
•
O,
0...
0,9
0,;.
09
0
""
<6-... ""1
..."" iS'
>
"%,...~
""1iS'.
~
-j,
...",,'10' •
"'10''";.
•
••
•
"0
••
•
•
"'0
~
!J'
<:
Z
<:
<:
lI:
lI:
~
~
z
:>
•
z
:>
ë$
i!>"E!
~.g
a"-a
OUl
•
.66-
,
c9/~0
0.(:
~~'1Q1 •
'YQ) 0"1 0
~~
\S'I.
0"1 '"
.--.
\S'I~
<<$-
0.(:
~~
\
'YQ) ~
~~
\S'I.
0"1 -}
\S'l,
~
6-
ct:>.
.s><$- ~
~/ct:>.'1Q1"
~ ~~ '1Q1.
~ 0- 0"1 'Y
0Jl ~~ \S'I b
...
1
"
. . . ~.
.. '
.'
Il
•
0.(: &~~ '1Q1.
.
'YQ) 0"1 ~ •
~~ \S'I.
0"1 ~ fit
\S'l'
~
1J
11
~ 4
11
6'0
~~
CON
........
c..,e.
~Bi
0
......
CO
III
1
(
1
,
,,
•
>i
fi;
11
.... 0)
ve»
BiC.
1
Plc.
1
~c.
'\~
-
1
,...
....
c.
'!?
D.
D.
•
•
•
"'~ &
~ ~
~
<QX' ()" ()" Q;-~ ~r" ~'If ()'~ r:<J(jv Or" (jv (jv Or" (jv ,(jv Or"
B
n'" '" n'" n'" ... '" n'" ...
~~~~~~~~
~'
Figure 5. Western analysis of
viral proteins. Sucrose cushion
purified viral particles were lysed
with RIPA buffer, and aliquots of
viral pro teins were resolved using
15% SDS-PAGE as previously
described(5).
Sera from human AIDS
patients and l25I-protein A/G(ICN)
were used to detect viral proteins.
Il.
<Q'
0' (),
),
V
~'
~,
Pr160!gp16<>;
gp120'
p66-
-
Pr55!p51gp41p39-
p32-
p24-
p17-
-
-
•
I!:I
.. ·II~I_."
-
•
3.4. Results
For each mutant studied, we have compared ta wild-type HIV-I: 1)
the 2L PAGE patterns of viral tRNA (Figure 2 ); 2) the relative amounts
of viral tRNALys3 incorporai:ed in the virus (Figures 3,4); and 3)
western blot viral protein patterns(Figures SA,Sa).
the
1be relative amount
of tRNALys3 incorporated/virus (relative ta wild type)
is determined by
measuring the amount of tRNALys3 and genomic RNA in a given viral RNA
sample, by hybridizing the sample with DNA probes specifie for either
tRNALys3 or genomic RNA.
probed with
Western blot patterns of viral protein~ are
anti-HIV sera.
Effect of carboxy deletions of pr160".,,-pol upon selective tRNALy03
incorporation and viral precursor processing.
We have constructed an integrase (IN)
BspMI,
deletion mutant,
SVC21
which expresses a truncated Pr160g ag -pol precursor prote!n which
lacks 90% of IN sequence.
The expression of this truncated Pr160 0s ,,-pol
termina tes with a novel stop codon inserted immediately after amino acid
30 of the IN sequence(Figure 1).
Transfection of this mutant proviraJ
DNA into COS-7 cells produces non infectious viral particles with wild
type levels of p24 and RT activity(Table 2). The production of viral
particles is expected,
sinee the COS cel1 transfection system bypasses
the reverse transcription and integration steps of the HIV-1 life cycle.
The 2D PAGE pattern of viral tRNA in the mutant
virus(Figu~e
2, panel E)
indicates a somewhat higher background of low molecular weight RNA than
is found for
the wild type viral
tRNA pattern (Figure 2,
panel Al.
Hybridization analysis of viral RNA fram SVC21 BspMI viral particles
reveals
that
for
equal amounts of genomic viral
RNA,
these viral
particles contain 64% of the tRNALys3 Molecules found in wild-type virus
(Figures 3, 4, and Table 3).
Western blot analysis(Figure SA, lane E)
shows primarily a wild type protein pattern, including bath RT(pSl/p66)
and CA(p24),
but the integrase band(p32)
is missing.
This protein
pattern indicates that bath the PrSS"so and the truncated Prl600so-pol
•
were incorporated into viral particles during viral assembly.
These
data indicate that the removal of integrase sequence in Pr1600s 0-pol does
143
•
nct
affect
either
selective
tRNALys
packaging
Pr160g a g-pol
or
incorporation inta HIV-l
On the other hand, deletions extending inta the carboxy portion of
sequences cading for RT eliminate selective tRNALys3 packaging.
one shows the DNA position at which each deletion begins,
i.e.
Table
DrdI and
SVC21 DrdII are both de1eted from nucleotide position 3586, while the
mutant SVC21 RdI deletion begins at position 3586.
The deletions are
out of frame. and, as described in experimental procedure,
the actual
truncated Pr160gag-pol sequence is extended beyond the deletion start
point 35,
16,
and 1 non-Pr160 Qag -pol ami no acids befo:-e reaching new
termination codons.
Transfection of these mutant proviral DNAs inta
COS-7 cells produces viral particles containing truncated Pr160gag-pol
lacking part of the connection domain, and aIl of the RNase H domain and
integrase sequence(Figure 1).
Transfections
yielded
non-infectious
viral
particles
with
background levels of RT activity(Table 2). Analysis of the tRNALys3
content in SVC21 DrdI, SVC21 DrdII, and SVC21 RdI virus(Figures 3, 4,
and Table 3) showed non-selective incorporation (respectively, 16%, 14%,
and 14% of wild type \liraI tRNALys3 content).
Similarly,
the 2D PAGE
patterns of tRNA in these virus(Figure 2, panels B-D) indicates nonse~ective
tRNALys incorporation.
Western blot analysis(Figure SB, lanes B-D) shows the absence of
RT and
integrase bands,
cleavage product, p39.
and
the presence of
the
intermediate Gag
The presence of p39 sequences can be seen when
insufficient functional protease(PR)
is present in the virus(l, 41).
This is also reflected in the unusually high ratio of Pr55 gag to p24
seen in these virus. This incomplete Pr55g a g processing could be due
either
to
inefficient
Gag-Pol
incorporation
and/or
inefficient
proteolytic processing of the mutant Pr160gag-pol precursor.
Mutationa in thll fingllra,
palm,
and thUlllb domaina in HIV-l Ri' ahow
littlll or no affllct upon alllllctivll tRNALya incorporation and prllcuraor
procllaaing.
•
X-ray crystal10graphy studies of RT
the
fingers,
domains(24).
palm,
thumb,
connection,
resolved the
and
RNase
Molecule into
H
structural
The linear 10calization of these domains in RT is shown in
144
•
Figure l, and we have tested the affect of mutations in these ragions on
tRNALys3 incorporation into the virus and precursor protein processing.
For exarnple,
we have constructed amine acid insertion mutants
fingers(SVC2l R3a & SVC2l R3b),
domains of RT.
palm(SVC2l R6),
in the
and thumb(SVC2l A2a)
Viral particles produced from transfection of these
mutant proviral ONAs into COS-7 cell contained CA(p24), RT(pSl/p66) and
IN(p32)
sequences,
an
indication
PrSS gag
of
incorporation and processing(Figure SA,
lanes F-I)
particles also have a reduced RT activity
These mutant viral
(Table 2).
mutants had 17% to 63% of wi:d type RT activity,
Pr160 gag -po!
and
Although these
their abilities to
infect MT4 cells dropped even lower, varying between 0.13% and 16.38% of
wild
type
infectivity.
This
may
indicate
that
the
artificial
polyrA/oligodT primer template used in the RT assay is less sensitive to
mutational defects in RT than in vivo reverse transcription. or that the
mutations may have additional replication defects besides the inhibition
of RNA dependent ONA polymerase activity.
Although SVC2l R3a and SVC2l R3b both contain a six nucleotide
insertion at amine acid 68 of the RT sequence, SVC2l R3b consistently
showed
a
somewhat
R3a(Table 2).
higher
RT
activity
and
infectivity
than
SVC2l
This may be because the less rigid amine acid side
chains(Table 1) present in the mutation of SVC2l R3b allowed the mutant
RT to fold into a conformation closer to that of the native RT sequence.
SVC2l R6 and SVC2l A2a have the same insertions, but these mutations are
located at either the palm-thumb junction,
thumb(aH) of RT,
respectively(17).
(in the
palm(~14))
or in
The ami no acid insertion in SVC2l
R6 consistently produces a greater degree of inhibition of RT activity
and infectivity than the identical insertion in SVC2l A2a(Table 2).
The tRNA from these mutant viruses produces wild
patterns when ana1yzed by 20 PAGE(Figure 2,
type
panels F-Il,
and these
viruses also contain wild-type amounts of tRNALya3
(Figures 3,
Table 3).
fact
These
results are consistent with the
tRNALya
4, and
that aU
H·IV
mutations found thus far which con tribu te resistance to inhibitors of RT
are found in the fingers and palm domains of RT(12,
•
resistance
mutants
must
assemb1y in order for
incorporate
primer
them to propagate,
145
39).
tRNALya3
Since these
during
viral
those resu1ts support our
•
findings that the fingers and the palm region of the RT are not likely
to be involved in selective tRNALYD packaging during viral assembly.
Tbe wild-type connection domain sequence is important for selective
tRNALy· packaging and precursor protein processing
Since the deletion mutations indicate that regions in the carboxy
part of the RT are important for obtaining selective tRNALys3 packaging,
we constructed six additional ami no acid insertion mutants (SVC21 H2,
SVC21 R7, SVC21 Ha4, SVC21 Dr2, SVC21 Ra, and SVC21 A4)and one amine
acid replacement mutant(SVC21 GR), carrying mutations in the connection
domain of RT, in the carboxyl end of the thumb, and in the amino end of
the RNase H domain (Figure 1).
Transfection of these mutant proviral
DNAs into Cos-7 cells yielded non-infectious viral particles
with
background levels of RT activity, with the exception of SVC21 R7 and
SVC21 A4, which show slightly elevated levels of RT activity(Table 2).
2D-PAGE of viral RNA sampI es from SVC21 R7, SVC21 Ra, and SVC21
A4, produced wild type patterns(Figure 2, panels K, N, 0), and these
virus contained 99%, 66%, and 103%, respectively, of the wild type viral
content of tRNALys3(Figures 3, 4, and Table 3). This suggests that the
regions at the beginning of the connection domain
end of the connection domain
RNase H domain (SVC2l A4)
packaging.
~22
(SVC21 Ra),
~lS(SVC2l
R7), at the
and at the beginning cf
are not essential
for selective tRNALYD3
However, analysis of viral RNA samples, SVC2l H2, SVC2l Ha4,
SVC:: Dr2, and SVC21 GR revealed non-selective tRNA incorporation in
vir"l particles (Figure 2, panels J, L, M, P).
These viral particles
contained only la%, 19%, 17%, and 21% of wild type tRNALYD3 per equal
amount of genomic RNA(Figure 3, 4, and Table 3).
Western blot analysis of the proteins in these mutant viruses
revealed a correlation between the presence of processed proteins and
the ability to selectively incorporate tRNALys3 into the virus. As shown
in Figure SA,
•
lanes K and 0,
SVC21 R7 and SVC21
A~
show wild-type
protein patterns, demonstrating p66(RT), p32 (integrase), und a high
ratio of p24/PrSS gag . SVC21 R8(Figure SA, lane N), while showing a high
p24/PrSS gag ratio, does not show p66 RT, "nd shows very little p32 IN,
which may imply a problem with processing of these products from
Pr160 ga g-pol. As noted in Table 3, SVC21 Ra shows a somewhat decreased
146
•
tRNA Lys3 incorporation.
GR virus (Figure SB,
The SVC21 H2, SVC21 Ha4, SVC21 Dr2, and SVC21
lanes J,
L,
M,
P),
which do not show tRNALys3
selective incorporation, show a complete absence of
These mutant viral particles show
well,
p66 RT and p32 IN.
incomplete Pr5S0so processing as
as demonstrat,d by the low p24/Pr550s 0 ratio and/or the strong
presence of the PrSS gag intermediate, p39.
precursor processing
These patterns of incomplete
show a strong resemblance te the protein patterns
obtained from virus which lack a functional Pr1600so-po 1 protease(l, 41).
SVC21 Dr2, which lacks p66 RT and p32 IN bands, does have less aberrant
processing of Pr5S0so , as shown by a higher p24/PrSSOs o ratio, although
p39 is still
~
prominent species.
Thus, the sarne mutations within and
flanking the connection domain which affect tRNALys3 incorporation also
appear to affect viral protein maturation .
•
147
•
Using a
transient expression system to express wild-type and
mutant proviral DNAs, we have found that deletion of the IN sequence and
amino acid insertion or replacement mutations in the fingers, palm, or
RNase
H
domains
of
RT
did
not
affect
either
the
selective
incorporation of tRNALys3 into viral particles or PrSS gag and Pr160 ga g-pol
processing. Carboxy deletions extending into the connection domain of RT
and smaller ami no acid mutations within and flanking the connection
domain of RT inhibit selective tRNALys3 incorporation and alter viral
precursor prote in processing.
Examining the ami no acid insertions and
replacements within the RT domain, we find that three of four mutations
which maximally affect tRNALys3 selection are found centrally in the
connection domain. These are SVC21 Ha4, SVC21 Dr2 and SVC21 GR.
wutation just 3' of these mutations, SVC21
R~,
The
shows an intermediate
inhibition of tRNALys3 incorporation, while the mutation further 3', in
the RNase H domain, SVC21 A4, shows wild type tRNALys3 incorporation.
The mutatio,,_ jusl S'of SVC21 Ha4"
SVC21 R7,
shows
wild type
incorporation of tRNALYS, as do most of the other mutations S'of this
region, with one major exception-SVC21 H2.
SVC21 H2, just S'of SV21 R7
at the thumb/connection domain junction, shows maximal inhibition of
tRNALysJ incorporation.
The four mutant viruses which show maximum inhibition of tRNALys
incorporation also show the greatest absence of cleaved precursor
products in the virus. One of these, SVC21 Ha4, shows
~nly
background
levels of RT activity(Table 2), but it has been shown to have wild-type
RT
ac~ivity
vitro(34).
when the mature RT is expressed in a bacterial lysate in
This indicates that the low level of RT activity we obtain
for this mutant is not a result of non-functional RT, but rather due to
th~
lack of processed RT sequences appearing in the viral supernatant.
In this work, however, we have not ascertained for this mutant, or the
other three mutants,
•
wheth"r this is due to Pr160gag-pol not being
efficiently packaged into the '-rirus, or to the inefficient release of
viral protease from the mutant Pr160 gag-pol. The polyclonal antibody used
for
the Western blots detects a 1;;:,nd ut the position whp.re both
Pr160g ag-pol and gp160 en,·plope protein migrate. While our attempts to
148
•
detect Pr160 gsg -pol with anti-RT monoclonal Ab have thus far yielded
ambiguous results, our data indicate that the presence of the 160kd band
correlates very well with the presence of processed PrSS gsg and Pr160 gsg pol products in the virus.
For example the 160kd species is present in
aIl virus represented in Figure SA, but in Figure SB, aside fram its
presence in wild-type virus, is only detected in th3 SVC21 Dr2 virus,
the only mutant virus in that figure which shows sorne degree of PrSS gsg
processing (In this virus ,as weIl as aIl others shown in FiguL'e SB,
neither p32 IN nor p66 RT are detected).
It is therefore possible that
tRNALys3 selective packaging correlates with packaging of Pr160gSg-pol,
i.e., connection demain mutants interfere with Pr160gag-pol incorporation
into the virus.
effects
of
We are current1y attempting to directly measure th~
the connection domain mutations upon Pr160g s g- pol
ir.corporation into virus by inserting the mutations studied herein into
a protease-negative proviral DNA.
The dependence of Pr160gsg-pol packaging on the myristylation of
PrSSgag(30, 46) suggests a model where Pr160g s g-pol might be carried to
the membrane by interaction of its amino-terminal portion with the
homologous sequences in PrSS gSg .
SequGnces in the carboxy portion of
Pr160gsg-pol could interfere with this interaction in several ways. The
Pr160 gs g-pol conformation might be altered, which might prevent a
required homodimerization, or prevent interaction with PrSS gsO , Both
the leucine
zipper-lik~
region and the tryptophan repeat motif have been
shown to play a role in protein-protein interactions (7, 38) , and both
these structures are found in the connection damain. Conformational
change might also affect Pr160gsg-pol
~ecenL
interact~on
with cellular proteins.
studies have shown that cellular pro teins internct with viral
components at various stages of the retroviral life cycle(ll, 16, 21,
23, 47, 49), and
~-actin
has recently been reported to bind to mature RT
and the Pol precursor protein in vitro (16) . The removal of IN sequeloces
might
~ave
the least effect upon precursor conformation because it is at
the very terminus of the molecule. The fact that mutations within the IN
sequences have been found to impede Pr160"sg-pol packaging (8) may then
•
imply a negative effect of the altered sequences present upon the more
internal conformational domains of the molecule .
149
•
Another possibility
connection damain
because
of
is
lt
flanking
the
prE'/ent tRNALys .tram binding te Pr160ge:~-r'ol, ei ther
conformational
changes
elsewhere in the rnolecule, or
mutation.
that mutations within or
affecting
beca~tse
has been reported
a
tRNA
binding
site
the binding site is close ta the
that the interaction of tRNALYs with
mature RT induces conformational changes(40). Tne 3 amine acid insertion
of SVC2l H2
is found in
the C-terminal end of R'l'-I;humb domain(aJ),
whict. is in close proximity with the helix clamp strncture in the thumb
domain.
The helix clamp structure has been implicated to be a nucleic
acid binding motif (4,
14) .The connection domain of
the p5l has been
suggested to be the primer/template binding pocket of the RT(24).
SVC2l
Ha4 and SVC2l Dr2 contain ami no acid insertions in the ~17 and the ~19
of the connection damain, and the SVC21 GR mutation replaces a conserved
tryptophan repeat sequence found in the aL to
~20
of the connection
domain(The functional significance of the tryptophan repeat sequence is
implied by the fact that it is conserved in various strains of HIV-l,
HIV-2, and SIV (2».
There lS aIse evidence, fram a crosslinking study performed on a
synthetic tRNALys3 / h~V-l RT complex(28),
have found te affect tRNALys3
when mutated also
interaction
of
that regions in RT whicl.l we
incorporation and precursor processing
interact directly with tRNA Lys3.
tRNALys3 was
found
No evidence for
for CNBr RT fragments
primarily the fingers or palm domai.,s.
On the other hand,
containing
a carbnxy
terminal p66 fragme::" (aa residues 357-560) con'"aining the carboxy half
of the connection domain and the RNase H domain interacted with the 5'
t·rminus of tRNALys3, while fragments from both p66 and p51(representing
aa
residues 230-356)
cont"i~ing
the thumb and amine portion of
the
connection domain, ward found to interact with U36 in the anticodon of
ti<I.ALys3.
Although
their work deals with mature
synthetic tRNALys3 interaction in vitro,
RT and unmodified
their results match our own
regarding the regions in the RT sequences of Pr160gs g- pol which inhibit
tRNALys3 incorporation when mutated. These observations suggest that our
mutations may inhibit selective tRNALys3
•
incorporation by preventing
stable interaction wi th tRNALys3 molecules.
It is also possible that
the interactions of tRNALys with Pr160g s g-pol might additionally enhance
150
•
the pack~ging efficiency of Pr16ogag-pol by altering its confor.mation and
stabilizing its interaction with other molecules such as Pr55g ao.
While mutations in the RT sequences of avian virus have also baen
found to prevent primer tRNA incorporation (32) , results contrary to this
have been reported using MuLV(26, 26b).
Levin and her colleagues found
that removal of extensive carboxy-terminal regions in the RT of MuLV did
nct affect either the inc~rporaticn or placement
in this virus.
The MuLV
~utant
resulted in the removal of
th~
~tudied
of the primer tRNAPro
was a frame-shift mutation which
last part of a region
enalog~us
to the
p66 ccnnection dama in of HIV RT, all of RNase H, and al1 of integrase.
These contralY results may be due to the different structures of HIV-l
and MuLV RT's.
For example, MuLV RT is monomeric while HIV-l and ALV
RTs are heterodimeric. HIV(3,
43) and ALV(6,
13) have been shown to
selectively interact in vitro with their primer tRNAs (tRNALys3 and
tRNATrp,
respectively),
wh:ile MuLV RT does not appear capable of
selectively interacting with tRNAPro. (29) .
•
151
•
3.6. Acknowledgment
This work was supported in part by grants from the National Health
Research Development Program, Health and Welfare Canada, and the Medical
Research Council(Canada).
We
thank Sandy Fraiberg
for
assistance
rnanuscript .
•
152
in preparation
of
the
•
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159
•
Preface(Chapter 4)
Ancther aspect of viral tRNA in the HIV-1 replication cycle is the
genomic placement of primer tRNALys3 in vivo, which i5 essential for the
efficient reverse transcription of viral cDNA.
In this chapter, we
provide preliminary data ta show that the processing of the
~ill
pr~cursor
pro teins and the presence of integrase sequence are nct required for the
genomic placement oi primer tRNALys3 ta oceur in vivo.
studies
show
that
a
viral mutant,
SVC21
GR,
which
Primer extension
does
not
show
selective tRNALys incorporation into viral particles, also shows reduced
genomic placement of primer tRNALys3 in vivo .
•
160
•
Chapte= 4
The Genomic Placement of Hrv-l Primer tRNALYB3 Occurs in the
Absence of a Functional Protease or Integrase Sequences
•
161
•
4.1. Ab.tract
The placement of tRNALys3 ante the PBS of HIV tas been detected ry
measuring the ability of in vivo placed tRNALys3 to be extended in an in
vitro
RT
reaction.
primer/template.
Total
viral
RNA
was
used
as
the
source
of
We have examined the effects of Pr160ga g- pol mutations
on genomic placement
in
HIV-l.
1)
Virus
containing
an
inactive
protease, and which therefore contain unprocessed PrSS gag and Pr160 gag pol, still show genomic placement of primer tRNALys3.
2) Virus lacking
90% of IN sequences still show both selective tRNALys3 packaging and
placement of the primer tRNALys3.
These two results indicate that
either Pr160gag-pol is not required for primer tRNALys3 placement, or if
it
is,
its
sequences
processing
~'re
by
protease
and
the
presence
of
integrase
nct required for its function in genomic placement.
The replacement of the conserved tryptophan repeat sequences
3)
in the
connection demain of the RT inhibits selective tRNALys3 incorporation,
and this mutant virus has a significantly reduced level of primer
tRNA Lys3 placement ante the genomic RNA.
This suggests several
alternative possibilities: 1) Select tRNA incorporation is a prerequiste
for genomic placement.
2)
Genomic placement does nct oceur because
Pr160gag-pol is not in the virus.
J)
The wild type tryptophan repeat
sequence "s necessary for the genomic placement of the HIV-l primer tRNA
in vivo.
Further experimentation will be required to choose from these
possibilities .
•
162
Figure 1. Structure of wild type and mutant HIV-l plasmids. Al SVC21 BH10
contains the wild type HIV-1 proviral DNA sequences. BI SVC21 P(-)
contains a protease-deficient HIV-1 proviral DNA due to a point mutation
at amine acid 25 in the protease region. Cl SVC21 BspMI is an integrase
deletion mutation, which lacks 90% of the integrase sequences from the
carboxyl terminal end.
D) SVC21 GR is a tryptophan repeat mutant. A
stretch of conserved tryptophan repeat sequences in HIV-1 RT connection
domain are replaced with glutamic acids as shown in the digram.
•
•
•
•
A, SVC21 BH10
PBS
1
U3
EBLJ
gag
I!
pol
~::;env
:I?@ ~3 ~
~~:;
~
:I?d
B, SVC21 P(-}
PBS
l
~s
U
Asp25-Arg25
gag
1
lL
pol
vpu
env
_
U3
~
1
C, SVC21 BspMJ:
PBS
1 U3
sl
EBLJ
gag
1p
b1
RT
-- ~::; env
Il!EJ
:P@U3
vpu
naf
1
~
D, SVC21 GR
PBS
U3
gag
vif
.T
rav
,r
env
vpu
wild type
GlnLysGluThrTrp GluThrTrpTrp ThrGluTyrTrp GlnAlaThrTrp lIeProGluTrp GluPheValAsn
GR
GlnLysGluThrGlu GluThrGluGlu ThrGluTyrGlu GlnAlaThrGlu lIeProGluGlu GluPheValAsn
U3 IllIu
Figure 2. Schematic representation of the in vitro RT assay.
Using total viral RNA as the source of primer/template,
a- 32 p-dGTP, ddATP, dTTP, and dCTP are mixed with total viral
RNA, RT buffer, and exogenous RT.
RT polymerization is carried
out at 37°C for l hour. The primer tRNA is labeled by the
presence of a- 32 p-dGTP, and the reaction is terminated by the
incorporation of ddATP
•
•
•
•
Primer Extension Assay
t:-~
0
f....~
§J
A..,q,
~J>~
,. fo"q,
'"
f.l.q,~
'!l
ACC
'
~.TGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACCTGAAAG~
*
ddATCGTCACC
'
~TGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACCTGAAAG~
•
A
B
~n
, - - - - - - -_ _-,
•••
Viral RNA
o
••••
Molecules of
Standard RNA
B
~,O
1.5
100
lB
RNA mnlecules (lOQ)
c
pr:mer Extension
•
...... 1
Figure 3. Primer extension of wild t}~e and mutant
viral RNA. A. Dot blots of ~ither known volumes of
viral RNA samples or known amounts of in vitro
transcribed genomic RNA are used for hybridization.
The blots are hybridized with a DNA oligomer(790H)
complementary to HIV-1 genomic RNA sequence 338354 (DNA sequence 791-807) . B. The relative
intensities of hybridization signa1s of in vitro
transcribed RNA were used to plot a standard curve.
Concentrations of genomic RNA from the viral RNA
samples were calculated from this curve. C.
Equivalent amounts of genomic RNA in total viral RNA
were used for primer extension ana1ysis, and rifferent
concentrations of wild type viral RNA were used as a
standard
•
4.2. Resulta and Discussion
In retrovirus, a hast cell derived tRNA is used as the primer for
th., RT-catalyzed synthesis of retroviral minus-strand DNA(14).
The 3'
terminal 18 nucleotides of the primer tRNA i3 complementary te a ragian
near
the 5'
end of
binding site(PBS).
the 35 S RNA genome which is
Other tRNAs(tRNAPro,
have
as
been
the primer
The PBS sequence in HIV-1 suggests that tRNALys3 is
the primer tRNA for HIV-1(12).
aIse
termed
shown
te
act
the
retroviruses(for review, see (G)}.
natural
tRNATrp,
primers
tRNA Lysl.2)
in
different
Primer tRNA incorporation into the
retroviruses i5 selective(S, 8, 10), and rernoval of RT sequences in the
retroviral
proviral DNA
inhibits
selective
tRNA packaging
into
the
virus(?, 10, 11).
The expression of PrSSoaO{the viral structural protein precursor)
provides sufficient information for viral particle formation and release
from
the
host
requires
the
cell
membrane (15) ,
expression
of
both
enzymatic protein precursor) (10) .
but
~"lective
tRNA
incorporation
Pr55g s g and Pr160g sg -pol (the
viral
We have previously shown that
the
selective tRNA incorporation into HIV-1 is independent of the packaging
of qenomic RNA,
and the proteolytic cleavage of the precursor proteins
is not necessary for the selective packaging of primer tRNA Lys3(10).
We
have aIse found that the RT connection demain in the Gag-Pol precursor
could be involved in the selective incorporation of primer tRNALys3 into
HIV-l (9) •
In vitro,
and
RT
genome.
is
RT has been shown to specifically bind to tRNALys3 (1),
thought
to
facilitate
Since Pr160g s g-pol
the placement of
tRNALys3
onto
the
is involved in the selective packaging of
primer tRNAw s3 , it is possible that the Pr160 gs g-pol is also involved il1
the genomic placement of primer tRNALys3 in vivo.
report
th.. following:
1)
In this work, we
We have measured the genomic placement of
primer tRNALysJ in a virus containing an inactive protease, i.e. p~ssgag
and Pr160gsg-pol are not processed.
We find that the genomic placement
of primer tRNALys3 is inciependent of precursor protein processing.
•
Pr160 ga g-pol
precursor
genomic placement,
protein may be
involved
in
Thus
the mechanism of
since pr160g ag-pol cantains RT s€CJuences which are
likely to be involved in the genomic placement of tRNALys3 in vivo.
168
2)
•
We also
show that
the absence of
integrase sequences aise do es
affect the gp:lomic placement of primer tRNALys3 in vivo.
not
\~e show
3)
that in a mutant virus containing mutations within the connection donulin
of RT, genomic placement of tRNALys3 is reduced.
cell~
Virus were produced fram transfection of COS-7
type
or mutant
proviral
transfection method.
DNAs (Figure
1)
using
the
with wild
calcium phosphtlte
Wild type and mutant virus were collected and were
purified by ultra centrifugation cnta a sucrase cushion,
and wild type
and mutant viral RNAs were extracted for RNA analysis as previously
described(9).
We have used an
in vitro primer extension Assay ta
examine
the efficiency of primer tRNA placemert. onto
genorne.
In this Assay, equivalent amounts of genomic RNA in total viral
RNA were used(Figure 3A
mutant
viral
RNAs
3B).
&
were
The concentration of wild
quantified
by
dot
blot
were used for each primer extension reaction.
reactions were prepared by mixing
pH 7.5,
200mM dCTP,
60mM KC1,
type and
hybridization
as
The primer extension
the viral RNA wi th RT buf fer (SOmM
3mM MgC12,
10mM DTT), dNTP mix(200mM dT'rp,
a- 32 P-dGTP (specifie activity of
S~Ci of
and 50mM ddATP) ,
3000Ci/mmol),
RNA
Approximate 5 x 10 8 copies of genomic RNAs
previously described(9).
Tris-HC1,
the HIV-l
20 units of the RNase inhibitor, RNAsin(Promega), SOng of
purified HIV-l RT, with a final reaction volume of
20~1.
Reactions were
allowed ta proceed at 37°C for one hour, and the reactions were phenol
extracted and the extended primers were precipitated along with
cold bacteria tRNA a,:: ":;c:arrier.
dried,
EDTA,
and were mixed with
0.05%
Bromophenol
S~l
Blue,
The precipitated viral RNAs
of loading buffer(9S.
0.005%
3~g
WArè
iormamide,
Xylene Cynaol).
of
air
20mM
The extended
primers were separated from the genome by heating the samples to 90°C
for
3
min,
and
electrophotesed
incorporated
a
quick
using
into
the
6%
co01in9
on
1ce.
denaturing
viral
cDNA
are,
The
PAGE.
samples
The
sequentially,
first
were
6
CTGCTA.
then
bases
The
presence of ddATP will therefore terminate the polymerization reaction
six bases 3'
of
the reverse transcription start site.
primer was labeled using radioactive a- 32 p-dGTP(Figure 2).
•
The extended
This assay
allows quantit~tion of the primer tRNALys3 that has already been placed
onto the PBS sequence of the genomic RNA in vivo .
169
•
Three concentrations of wild type viral RNA(lO",
were used as standards for the primer extension assay.
50 .. and 100")
Transfection of
SVC21 P(-) proviral DNA into COs-7 cells yields immature virus with
electron-lucent
viral
core
structure(4).
These
inunature
protease-
negative virus show selective packaging of the primer tRNALys3(10).
Although they package genomic RNA as efficiently as
virus(2,
10),
ta have a
dimer,
the wild type
the dirneric RNA of protease-negative HIV-l has been shown
less stable dimer structure than the wild type genomic RNA
and it has been suggested that viral genornic RNA undergoes a
protease-dependent maturation pro cess ta ferro a thermally stable dimer.
Our
primer
E..<tension
study
shows
that
the
efficiency
of
genomic
placement of the primer tRNALys3 in SVC21 P(-, is similar to that of the
wild type virus(Figure 3).
These data show that the genomic placement
of primer tRNALys3 in HIV-l is independent of Pr55g ag and PrlSogag-pol
processing, and that the genomic placement of primer tRNALys3 in wild
tY(.le virus cao ooeur prier ta this processing and te the maturation of
viral RNA dimers.
SVC21
BspMI
virus
contain
a
truncated
Pr160gag-pol
protein which terminates, as a result of a novel stop codon,
precursor
immediately
after amine acid 30 of the IN sequence, which represents only 10% of the
wild type IN sequences.
SVC21 BspMI virus has been shown to demonstrate
selective tRNA packaging, and they conta in approximately 60 .. of thA
tRNA Lys3 molecules found in wild type virus. Western analysis of SVC21
BspMI
virus
show
normal
processing
of
precursor
expected absence of mature IN sequences (9) .
proteins
In Figure 3,
wi th
the
we see that
the primer extension analysis of SVC21 BspMI viral RNA shows that the
efficiency of primer tRNALys3 placement onto the aIV-l genome is similar
to that of the wild type virus, indicating that the integrase sequence
is not required for the
genomi~ ~lacement
of the HIV-l primer tRNALys3.
'l'ransfection of SVC21 GR proviral DNA(DNA containing mutations
into Trp repeat sequences of the connection domain in RT) has been shown
to yield viral particles not showing selective tRNALys3
incorporation.
SVC21 GR virus contain only 20% of tRNALys3 molecules found in wild type
HIV-l virus,
•
and precursor protein processing is inhibited.
Primer
extension studies of SVC21 GR viral RNA show a significant reduction of
HIV-l genomic placement of primer tRNALys3.
170
•
Earlier work
in aviar:. sarcoma-leukosis virus (ASLV)
showed that
protease-defective virus encapsidate normal arnounts of genomic RNA and
primer tRNATrp.
Although some of the ASLV genomic RNA in the
negative virus was
found
te
be monomeric,
primer tRNATrp was not affected(13).
dernonstrates
required
that processing of
for
the
genomic
retroviral gencme.
proteQ~e­
the genornic plèlcement
of
Our work in the HIV-l system also
the viral
placement
precursor proteins
of
primer
tRNALyuJ
is not
onta
the
The presence of rnonomeric genomic RNA in the ASLV
protease-negative virus probably results fram the dissociation of the
less
stable
genomic
retroviruses{2, 3).
RNA
dimers
found
in
protease-defective
In addition, the HIV-l IN sequences are dispensable
for the genornic placement of primer tRNALys3.
These results imply that
the genomic placement of retroviral primer tRNA occurs prior ta the
maturation
of
viral
and genomic
pro teins
RNA.
On
the other hand,
mutation of the tryptophan repeat sequences in the connection dama in of
RT
sequences
tRNALys3.
significantly
SVC21
processing
of
GR virus
have
PrSS gag
the
inhibits
and
genomic
been
a
shown
complete
placement
to
have
absence
of
an
ot
primer
incomplete
p66
reverse
transcriptase and p32 integrase in western blot, which may indicate an
inability of Prl60 gag -pol mutated in the tryptophan repeat sequence to be
incorporated
into
the virus
or of
the viral
plotease
ta
carry out
proteolytic processing of the viral precursors(9).
Theze results
mechanism
of
sugge~t
genomic
several alternative interpretations for the
placement
of
primer
tRNALyo3,
experiments are required bafora choosing among
packaging
of
tRNALys3
and
them.
the efficient genomic
and
additional
'l'he selective
placement
ot
primer
tRNALys3 in the absence of functional protease and integrase sequences
could irnply that Pr160gag-pol is not involved in genomic placement, and
reduction
of genomic placement
tRNALys3
in SVC21 GR mutant directly
reflects the non-selective packaging of tRNALys3 found in this mutant.
Alternatively,
•
Prl6ogag-pol may be involved .'n the genomic plac"ment ot
primer
tRNA Lys3,
simply
not
and functional protease and integrase sequences are
genomic
placement in SVC21 GR virus could result from the absence of Pr160 gag -pol
packaging,
required
or result
for
this
process.
The
inhibition
of
from a mutated Pr16ogag-pol that is inca,>ab1e to
place primer "RNALys3 onto the genome.
171
•
4.3. References
Barat,
1.
1989.
C.,
HIV-1
V. Lullien,
Reverse
o. schatz, G. Keith,
Transcriptase
Specifically
Anticodon domain of its Cognate Primer tRNA. EMBD J.
2.
Pu, W., R. J. Gorelick,
Human
and A. Rein.
Immunoùeficiency Virus Type
1994.
1 Oimeric RNA
and J.
Interacts
L.
Darlix.
with
the
8:3~79-3285.
Characterization of
from Wild-Type and
Protease-Oefective Virions. J. Viro1. 68:5013-5018.
3.
Pu,
W.,
and A. Rein.
1993.
Maturation of Oimeric Viral RNA of
Mo1oney Murine Leukemia Virus. J. Viro1. 67:5443-5449.
4.
G6ttlinger, H. G., J. G. Socl.roski, and W. A. Hassltine. 1989. Ro1e
of Caps id Precursor Processing and Myristoylation in Morphogenesis and
Infectivity of Human
Immu~cdoticiency
Virus Type 1. Proc. Natl. Acad.
Sei. USA. 86:5781-5.
5.
Jiang, M., J. Mak, A. Lacl.ha, E. Cohen, M. Klein, B. Rovinski, and
L. Kleiman. 1993.
Id.,ntification of tRNAs Incorporated into Wild-Type
and Mutant Human Immunodeficiency Virus Type 1. J. Viro1. 67:3246-3253.
6.
Leis, J., A. Aiyar, and D. Cobrinik. 1993. Regulation of Initation
of Reverse Transcription of retroviruses, p.
S.
P.
Goff
(ed.),
Reverse Transcriptase,
33-~7.
vol.
1.
In A. M. Skalka and
Co1d Spring Harbor
Laboratory Press, New York, NY.
7.
Levin,
J.
G.,
and J.
G.
Ssicl.man. 1981. Effect of Po1yrnerase
Mutations on Packaging of Primer tRNA Pro during Murine Leukernia Virus
Assemb1y. J. Viro1. 38:403-408.
8.
Levin, J. G., and J. G. Seicl.man. 1979. Selective Packaging of Host
tRNAs by Murine Leukemia Virus Particles Ooes Not Require Genomic RNA.
•
J. Virol. 29:328-335 .
172
•
9.
Mak,
Kleiman.
J.,
Q. cao,
1996.
Pr160Q'Ag-pol
l. Lowy. V. R.
Prasad, M. A. Wainberg,
and L•
Reverse Transcriptase Connection Domain Mutations
Inhibit
the Select
Incorporation of Primer tRNA Lys3
in
into
HlV-l. J. Virol. Submitted.
10.
Jian~,
Mak, J., M.
and L. Kleiman.
Incorporation
M. A.
Wainber~,
M.-L. Hammarskjold, D. Reko.h,
1994. Role of Pr160oAo-pol in Med,ating the Selective
of
tRNALys
into
Human
Immunodeficiencv
Virus
Type
1
Particles. J. Virol. 68:2065-2072.
11.
Petera, G. G., and J. Hu. 1980. Reverse Transcriptase as the Major
Determinant for Selective Packaging of tRNA's into Avian
SdrCOll\d
Virua
Particles. J. Virol. 36:692-700.
12.
S.
Ratner, L., W. A. Haseltine, R. Patarca, K. J. Livak, B. Staroich,
F.
Josephs,
E.
R.
Doran,
Baumeister, L. Ivanoff, S. R. J.
T. S. Papas, J. Ghrayeb, N. T.
J.
A.
Rafal.ki,
P8tt~waYI
Chan~,
B.
A.
Whitehorn.
K.
P. M.L., J. A. LautenbQrger,
R. C. Gallo, and F.
Won~-staal.
1985. Complete Nucleotide sequence of the AlOS Virus, HTL,V-lII. Nature.
313:277-284.
13.
Stewart, L., G. Schatz, and V. M.
Vo~t.
Retrovirus Particles Defective in Viral
1990. ProperLies of Avian
Protf=dse.
J.
Virol.
64: 5076-
50n.
14.
Waters, L. C., and B. C. Mullin. 1977. Transfer RNA in RNA Tumor
Viruses. Prog. Nuc1eic Acid Res. Mol. Biol. 20:131-160.
15.
wills, J. W., and R. C. Craven. 1991. Fo=m,
Retrovira1 Gag Proteins. AlOS. 5:639-654 .
•
173
Function, and
Us~
of
•
Chapter 5
General Discussion
•
174
•
5.1. General Discussion
In this work, we have examined the mechanlsm of selective tRNALys
packaging into HIV-l anà clone preliminary work on factors effecting the
genomic placement of tRN~Lys3.
While our work clarifies the importance
of Pr1600 0 0- po1 in tRNALyo packaging,
it also brings ta light important
unsolved questions about this understudied, yet essential process in the
retroviral life cycle.
We shall address sorne of these questions below.
It has bêen known for a long time that the viral packaged tRNAs
have a cellular origin(27).
Prior to our work in the HIV-l system,
Levin and Peters had independently shown the importance of RT in the
selective
packaging
respectively.
of
primer
tRNA
In the ASV system,
The
determined(18).
clone 23)
nature
of
the
MoMuLV(12)
and
ASV(18)
ASV particles lacking RT activity
demonstrated non-selective packaging
particles.
in
of primer
RT mutant
in
tRNA
this
into
the viral
system
was
not
In the MoMuLV system, two RT mutant cloneslclone 13 and
were examined (12) .
MoMuLV clone 13 does not contain any
detectable RT protein and RT activity, and these viral particles do not
show selective packaging of "irai tRNAs.
However, MoMuLV clone 23 is a
frameshift mutant, which yields viral particles containing a truncated
Gag-Pol precursoL lacking Lhe IN sequences and 40% of the RT sequences
from the carboxyl terminus, shows selective packaging of viral tRNAlll,
12).
These data suggest that the selective packaging of primer tRNA
into retroviral particles requires the presence of polymerase sequences
in ASV and MoMuLV, but that partial removal of carboxyl sequences in RT
can still allow for the selective viral tRNA packaging in MoMuLV.
In HIV-l, we provided evidence that Pr16000 0- po1 is involved in the
selective
incorporation
observed that a
of
tRNAL~lS
into viral particles.
We have
large deletion of RT sequences inhibits the selective
packaging of tRNALYs in HIV-l viral particles, which is consistent with
the above findings
in ASV and the MoMuLV(clone 13),
involved in the selective packaging of viral tRNAs.
Le.
that RT is
In addition, we
have shown that the protease-rnediated processing of precursor pro teins
•
is not
re~~ired
of the
PrSS goO
for the selective packaging of viral tRNAs.
Expression
precursor protein alone yields viral particles lacking
selective tRNALys packaging,
but expressing both the PrSS gog and the
17S
•
Pr160 ga g-pol
proteins
proteins(Vpr.
Vpu,
in
the
and Nef)
packaging of viral tRNAs.
evidence
for
the
absenc~
of
viral
Env
and
auxiliary
yields viral particles showing sel.:-ctivl."
This observation provides the fit'st direct
involvement
of
packaging of retroviral tRNAs.
Gag-Pol
Since
precursors
Pr55 0a g
expression
in
i5
selective
essential
for the formation of viral particles, we are unable ta eliminate the
possibility of an assisting role for Pr5S gao in the selective packaging
of tRNALYN into viral particles.
On the other hand, alteration(14) or
elimination(15) of the PBS, or inhibition of genomic RNA packaging(13,
15),
does
nct
affect
selective
tRNALys
packaging,
indicating
that
genomic RNA does nct play a raIe in this process.
It is nct known how many Pr160 ga g-pol molecules are found in a HIV1 viral particle.
The number of tRNALys rnolecules found per HIV-l virus
may reflect the nurnber of Gag-Pol molecules packaged into the virus.
We
have been the first to determine the nurnber of primer tRNA molecules
found in retroviral particles, and there are approximately 20 molecules
of tRNALys(12 molecules of tRNALysl,2 and 8 molecules of tRNALysJ)
per HIV-1 virus(8,
tRNA Lys3 results
rnolecules from 8
15).
found
Increasing the cytoplasmic concentration of
in an increase in the total number of viral tRNALyoJ
to
17 rnolecules per virus.
This
increase is by
li
corresponding decrease ef tRNALysl.2 fram 12 te 3 molecules per virus.
Thus the total nurnber of tRNALys molecules remains at 20 per virus(7).
This limitation in the quantity of encapsidated tRNALys could be due to
the available spacing in the viral tRNA compartment found inside of the
viral particles.
An alternbtive explanation could be that the number of
tRNALys molecules packaged reflects the nurnber of Pr160gag-pol molecules
incorporated into the virus.
Our mutational mapping of the HIV-l
Pr160g4g-po! shows that a
carboxyl deletion of IN sequences in Pr160g4g-po! does not significantly
affect selective tRNALys packaging, but carboxyl deletions which include
both the
IN sequence and the RNase H and connection domains of RT do.
Small amine acid insertions(2-3) placed into various domains of RT show
no effect upon tRNALys packaging when p1aced into the fingers,
•
palm,
part of the thurnb, and RNase H domains , but mutations within or just Nterminal of the connection domain inhibit selective tRNALya packaging .
A direct correlation has been found between these mutations which affect
176
•
lRNALyc] pacKaging and
the virus.
t~e
absence of mature Gag and Gag-Pol pro teins in
vlliether the mutations in the connection damain of RT inhibit
selective packaging of viral tRNA by directly blocking the primer tRNA
binding site(s)
in the Pr160gag-pol precursor or do
50
by lowering the
packaging efficiency of Pr160gag-pol inta the viral particles remains to
b(: determined.
It is thought that the packaging of Gag-Pol precursor relies on
the expression of myristylated Gag precursor proteins(16,
the
intermolecular
interactions
between
different
ddving particle formation and budding(2l, 29).
incorporat~d
21,
29),
molecules
of
with
Gag
Gag-Pol wouid then be
in ta the budding partiele through the interaction of its
amino-terminal domains with the identical on es in the Gag sequences.
This hypothesis implies that the Pol sequence would not be required for
Gag- Pol encapsidation,
and therefore,
mutations
in the Pol sequences
should not affect the packaging of Gag-Pol precursor.
However, we have
obtained evidence that rnay indicate the RT connection domain mutants
described herein may be inhibiting selective tRNA incorporation via the
inhibition of Gag-Pol packaging.
Supporting this premise,
it has been
fo':nd that the point mutation in the conserved zinc finger of HIV-l IN
sequences inhibits the packaging of Gag-Pol (5) .
rhe packaging of Gag-
Pol precursor pro teins may require the dimerization of Gag-Pol proteins.
AIl
the
Pol gene
products
have
been
shawn
ta
function
only after
dimerization(l, 4, 17, 19, 23), and the mutations that affect selective
packaging of viral tRN.a. are located in regions of RT that have been
shown to be important for RT dimerization(3, 20).
aiso require Gag/Gag-Pol
cellular
factors.
Gag-Pol packaging rnay
interaction, and/or Gag-Pol interaction with
A recent
report has
shown
the HIV-l
precursor interacts with the cytoskeletal protein
polyrnerase
P actin(G).
AlI of
these potential interactions with a Gag-Pol molecule rnay be affected by
the cannection domain mutations, and thê existence of
~uch
interactions
in the effects of connection damain mutations upon them is now being
studied.
The genomic placement of primer tRNA is affected by alteration in
•
both viral proteins and genomic RNA.
In ASV,
the genomic placement of
primer tRNATrp correlates with the selective packaging of primer tRNATrp .
An ASV mutant lacking RT activity and RT proteins demonstrates non-
177
•
selective tRNATrp packaging and ineffi=ient yenornic pl~c~ment at primel
tRNATrp(lB).
How12ver,
for
NoHuLV,
the
genomic
placement
ol
primer
tRNAPro occurs
in the absence of bath selective tRNAPro packllging (clone
13) (12) and in the presence of partial RT sequence(clone 23\ (12).
Our
own analysis shows that bath protease-defective and integrase-deletion
mutants
showing
selective
placement of tRNALY93.
tRNA
incorporation have wild
type genomic
The dispensable nature of protease funetion in
the genomic placement of primer tRNh has aise been reported in other
retrovirus,
virus,
i. e.
SVC21 GR,
significant
ASLV (22) .
However,
in a connection damain mutant
in which selective tRNALyn3 packaging is inhibited,
reduction
of
genomic
a
suggesL~
placement
occurs.
several alternative possibilities:
1)
tRNA incorporation 1s a
prerequisite for genomic placement.
2) Genomic placement does not occur
Select
because Pr160gag~pol is not in the virus.
repeat sequence is necessary for
primer tRNA in vivo.
This
3) The wild type tryptophan
the genomic placement of
the HIV-l
Further experimentation will be required ta choose
fram these possibilities.
The
genomic
RNA
sequence
is
aIse
important
placement of the retrovirai primer tRNA.
sequences
ta
observed
that
sequences
complementary
retroviral
reverse
for
the
genomic
By replacing the natural PBS
to
other
transcription
tRNAs,
it
prefers
natural primer tRNA for the synthesis of viral cDNA(2,
has
to
been
use
the
14, 25, 26, 28).
Evidence exists that this preference for using the natural primer tRNA
for reverse trancription 15 related to an additional non-paS interaction
occurring
between primer tRNA and genemic
primer/template
studies
suggest
that
an
RNA.
In
in
HIV-l,
interaction
vitro
between
the
anticodon loop of the primer tRNA and an A-rich loop upstream of the PBS
in the genome 15 required for the proper genomic placement of primer
tRNA in
vivo(9,
10).
The
importance of
this
interaction has
been
confirmed by a study in which both the PBS and the A-rich loop sequences
are replaced with sequences that are complementary to the 3'
tRNAHis and the anticodon loop of tRNAHis respectively(24).
I(His-AC)
•
reaches
infection,
still
kinetics
This HIV-
mutant possesses inita1 delay replication kinetics,
eventually
can be
wi1d
using
type
tRNAHis
replication
as
at
the stable primer.
partially explained by
178
kinetics
the possible
end of
day
but it
7
post
The de1ayed
'compensatory'
•
mutations found in thB genomic RNA
be
int~r8sting
mutant.
seq~ence
S'of the PBS(24).
It
wil~
ta analyzé the viral tRNA contents in the HIV-IIHis-AC)
An enhancement of ~RNAHin packaging will suggest that selective
packaging of tRNAHin is important for the wild type genomic placement of
primer tRNA,
while the absence of selective tRNA packaying suggest that
its not important for genomic placement .
•
179
•
5.2. References
1.
Darke, P. L., C.-T. Leu, o. L.J., J. C. Heimbach, R. E. Di.hl, W.
S. Hill, R. A. F. Dixon, and 1. S. Sigal. 1989.
Virus
Protease:
Bacterial
Expression
and
Human 1mmunodef\ciency
Characterization
of
the
Purified Aspartic Protease. J. Biol. Chem. 264:2307-2312.
2.
of
Cas, A. To, B. Klaver, and B. Berkhout. 1995. Reduced Replicdlion
Human
Irnmunodeficiency
Transcription
Primers
Virus
Other
Type
than
the
1
:1utants
Natural
That
Use
tRNALY93.
J.
HeVt:.'r-sl:'
Virol.
69:3090-3097.
3.
the
Divita, G., T. Reatle,
Dimerization
Process
of
and R. S. Goody. 1993. Characterization of
HIV-l
Reverse Transcriptase
Heterùdimer
Using Intrinsic Protein Fluorescence. FEaS Letter. 324:153-158.
4.
Enge1man, A., F. D. Buahman, and R. craigie. 1993.
of Discrete Functional Domains of HIV-l Integrase
a~d
Identification
Their Organization
within an Active Mu1timeric Comples. EMBO J. 12:3269-3275.
5.
Enge1man, A.,
G. Eng1und,
J. M. Orenatein, M. A.
~artin,
end R.
Craigie. 1995. Multiple Effects of Mutations in Human ImmunodeEiciency
Virus type 1 Integrase on Viral Replication. J. Virol. 69:2729-2736.
6.
Hottiger,
Schaffner,
M.,
and U.
K.
Gramatikoff,
Hubacher.
O.
Georgiev,
C.
Chaponnier,
W.
1995. The Large Sububit of HIV-1 Reverse
Transcriptase Interacts with ~-actin. Nucleic Acids Res. 23:736-741.
7.
1994.
Huang, L.-M., A. Joahi, R. Wi11ey, J. Oren.tein, and K.-T. Jeang.
Human
Immunodeficiency Viruses Regulated by Alternative trans-
activtors: Genetic Evidience for a Novel Non-transcriptional Function of
Tat in Virion Infecivity. EMBO J. 13:2886-2896.
•
8.
Huang, Y., J. Mak, Q. Cao, Z. Li, M. A. Wainberg, and L. K1eiman.
1994. Incorporation of Excess Wi1d Type and Mutant tRNA Ly.3 Into HIV-1 .
J. Viro1. 68:7676-7683.
180
•
c.
~.
Isel, C.,
1995.
Initation of Reverse Transcription of HIV-l: Secondary Structure
d
thé HIV-l
Ehr~Bmann,
RNA/tRNALy.,
G. Keith,
B. Ehresmann, and R. Marquet.
ITémplate/Primér)
Compléx.
J.
Mol.
Biol.
247,236-250.
la.
Ise1, C.,
R. Marquet,
1993.
Modified Nucleotides of
L,aup
Interaction
in
G. Keith,
the
C. Ehreamann,
tRNALys3
Modulate
Initiation
and B. Ehresmann.
Primer/Template Loop-
Complex
of
HIV-l
Reverse
Transcription. J. Biol. Chém. 268:25269-25272.
Il.
Levin, J. G., S. C. HU, A. Rein, L. l. Messer, and B. l. Gerwin.
1984.
Murine leukemia Virus Mutant with a
Transcriptase Cading Region:
Implications
Frameshift
in
the Reverse
for pol Gene Structure.
J.
Virol. 51:470-478.
12.
L..vin,
J.
G.,
and J.
G.
S ..idman. 1981. EUéct of Polymérasé
Mutations on Packaging of Primer tRNA Pro during Murine Leukemia Virus
Assémbly. J. Virol. 38:403-408.
13.
L..vin, J. G., and J. G. S..idman. 1979. Séléctivé Packaging of Host
tRNAs by Murine Leukemia Virus Particles Does Not Require Genomic RNA.
J. Virol. 29:328-335.
14.
Li, X., J. Mak, E. J. Arts, L. K1eiman, M. A. Wainb..rg, and M. A.
Parniak. 1994.
Effects of Alterations of Primer Binding Site Sequences
on HIV-l Réplication. J. Virol. 68:6198-6206.
15.
Mak, J., M. Jiang, M. A. Wainb..rg, M.-L. Hammarskjo1d, D. R..kosh,
and L. K1"iman.
Incorporation
1994.
of
Rolé of Pr160g·g-pol
tRNALYs
into
Human
in Médiating the Séléctivé
Immunodef iciency Virus
Type
1
Particlés. J. Virol. 68:2065-2072.
•
16.
Park, J., and C. D. Morrow. 1992. Tbé Nonmyristylated Pr160g·g-pol
Polyprotein
of
Human
Immunoc'.éficiency
181
Virus
Typé
l
Intéracts
with
•
Pr5S gag and is Incorporated into Viruslike Particles. J. Vir01. 66:6304
tS313.
17.
Pearl,
Retrovi~~l
18.
L. H.,
and W. R. Taylor. 1987. A Structural Model
t')l
th ...~
Proteases. Nature. 329:351-354.
Petera, G. G.,
Determinant
ana
J. Hu.
19130. Reverse Trar.scriptase
for Selective Packaging of
tRNA' s
dS
the Majnr
into Av idn Sarcomd Vi
nl~:
Particles. J. Virol. 36:692-700.
19.
Human
Restle,
T.,
B.
Muller,
lnununode f ie iency Virus
and R.
Type
S.
Caody.
1990.
Dimerization
1 Reverse Transer iptase.
J.
ot
Bi 0 1.
Chem. 265:8986-8988.
20.
Rastle, T., M. Pawlita, G. Sczakiel, B. Muller,
and R. S. Goody.
1992. Structure-Function Relationships of HIV-l Reverse
Tra~scriptas~
Determined Using Monoclonal Antibodies. J. Biol. Chem. 267:14654-14661.
21.
Smith,
A.
J.,
N.
srivivaBakumar,
!L-L.
HammarBlejllld,
and
O.
Rekosh. 1993. Requirernents for Incorporation of Pr160t,Jag-pol trom Human
lnununodeficiency Virus Type 1 Into Virus-Like
Particles . .1.
Virol.
67:2266-2275.
22.
Stewart, L., G. Schatz, and V. M. Vogt. 1990. Properties of Avian
Retrovirus Particles Defective in Viral Protease.
J.
Virol.
64:5076-
5092.
23.
Van Gent,
O. C.,
c.
Vink, A. A. M. Oude GroBneger,
Plasterk. 1993. Cornplementation between HIV
Int~grase
and R. H. A.
Proteins Mutated
in Different Domains. EMBO J. 12:3261-3267.
24.
Wakefield, J. K., B.-M. Kang, and C. O. Morrow. 1996. Construction
of a Type 1 Human Immunodeficiency Virus That Maintains a Primer Binding
•
Site Complementary to tRNA His . J. Viro1. 70:966-975 .
182
•
25.
Wakefield,
J.
F...,
H.
Rhim,
and
c.
o.
Morrow.
1994.
Minimal
SéquenCE.- Requirementz of a Functional Hllman Immunodeficiency Virus Type
1 Prim0r Binding Site. J. Viral. 68:1605-1614.
26.
Wakefield,
J.
K.,
A.
ImmunodcCiciency Virus Type
G.
Wolf,
and C.
D.
1 Can Use Different
Reverse Transcription but Selectively
Maintain~
Morrow.
tRNAs as
1995.
Human
Primers
for
a Primer Binding Site
Complementary to tRNALysJ. J. Virol. 69:6021-6029.
27.
Waters, L. C., Rnd B. C. Mullin. 1977. Transfer RNA in RNA Tumor
Viruses. Prog. Nucleic Acid Res. Mol. Biol. 20:131-160.
28.
Whitcomb,
Replication
J.
M.,
B.
A.
of Avian Leukosis
Binding Site:
Ortiz-Conde,
and S.
H.
Viruses with Mutations
Use of Alternative tRNAs as Primers.
Hughes.
at
the
J. Viral.
1995.
Primer
69:6228-
6238.
29.
wills, J. W., and R. C. Craven. 1991. Form. Function. and Use of
Retroviral Gag
~rcteins.
AIDS. 5:639-654 .
•
183
•
Chapter 6
Contribution of Original Knowledge
•
184
•
6.1. contribution to original Knowledge
Th~
following is
thesi~
Cl
summary list of rny original contributions fram the
to the scientific
comm~nity
under the supervision of Dr. Lawrence
Kleiman.
Chapt..r 2
We
were
tRNALys3
first
to
quantitatively
is selectively packaged
demonstrate
in ta HIV-l virus,
efficiency of tRNALysl.2 and tRNALys3 are similar.
isoacceptors
are
found
to
represent
6%
of
that
the
primer
and the packaging
tRNALysl,2 and tRNALys3
the
low rnolecule weight
cellular tRNA population, but it is found to be 60% of the total low
Molecule weight viral RNA.
The similar packaging efficiency between
tRNALy.l.2 and tRNALy.3 suggests that the viral packaging of tRNALy.l.2
and tRNALys3 utilized a similar mechanism.
2)
Neither the presence of
P8S sequences in the genomic RNA, nor the genomic RNA packaging inta the
virus,
is
required for
the selective packaging of tRNALys molecules,
this indicates that the mechanism vf genomic RNA packaging differs from
the mechanism of primer
tRNALys3 packaging.
determine
primer
average
the
eight
number
of
molecules
of
tRNA
tRNALys3
in
3)
We were the first
HIV-l (on average,
found
in
to
there are
each virus).
4)
We
demonstrated that the unprocessed Gag-Pol precursor is involved in the
selective packaging of viral tRNA, and the RT sequence probably has a
role in this process.
not sufficient
Gag
and
for
Gag-Pol
The expression of HIV-l Gag precursor alone is
the selective tRNALys packaging,
precursor
packaging of primer tRNALys.
protein
is
required
and expression of
for
the
selective
Inactivation of viral protease do es not
alter the selective packaging of primer tRNALy. Molecules, but an in
frame large deletion of the RT sequence inhibits the selective packaging
of primer tRNALy. Molecules into the viral particles.
Chapt..r 3
•
Mutant virus containing a truncated Gag-Pol precursor that lacks
IN sequences still show selective viral packaging of primer tRNALys3 and
185
•
or
...... ild t)''P1? processing
IN
sequences
15
not
pt"~C'u:-so!.·
!"t'quireà
St:>~lt.:t.>nct.>s.
pr..Jt02in
th~
t0r
selectivt:-'
5uggt'stin\.j th'lt
pdC"kd,::11ng
L't
pI'lnlt'f
tRNALyn3 and proteolytic cleavage of the vlrd: prote in pI-ècursurc.
1 RT is di"'ided into the fingers,
H domains.
toJhile
packaging,
a
palm,
IN sequences drt'
selective viral
ct
C"0nnectiùn.
dispensàblt>
carboxyl deletion which
the RNase H demain and
thumb.
tor
includes aIl ot
tiIV
dnd the RNrloSl'
selectlvt.·
the
thl.'
IN
tRN/\l.y:;
sequènct.~S,
portion of the connection dotndin ot RT inhibits
packaging
of
into the connection damain of
insertiLm~~
Small amino acids
tRNAl.yn3.
the RT or amino deid sequences
just
N~
terminus of the RT connection damain inhibits the selective pdckaging ot
primer
tRNALys3
finger,
into HIV-l virus.
but amino dcids insertions inta the
palm. part oE the thumb. and th€ RNase H demains oE RT does not
afEect the selective viral packaging eE primer tRNALyu3 inte the HIV-l.
A direct correlation is observed between mutations that aEfect tRNALyuJ
packaging ar.d the absence of mature Gag anà Gag-Pol
virus.
proteins
in
the
It is possible that mutations in the connection demain of RT
inhibits
selective
efficiency
of
the
tRNALys3
Gag-Pol
packaging
precursor
by
hindering
proteins.
and
the
packaging
that
selective
packaging oE tRNALysJ into HIV-l does not occur in the absence of CagPol protein packaging.
Chapter 4
From an in vitro RT assay that uses total viral RNA as the source
oE primer/ternplate. we have shown. that a protease deEective HIV-l which
contains unprocessed Gag and Gag-Pol
genomic placement of primer tRNA.
precursors still
has wild
type
This suggests that both viral protein
and genomic RNA maturation are not required for the genomic placement of
primer
tRNALysJ.
probably occurs
and that
the genornic placement of HIV-l primer tRNA
prior
the maturation of
to
the vir.3.1
core.
An
IN
deletion mutant virus containing a truncated Gag-Pol precursor lacking
90% of
the
IN sequence from the carboxyl
genomic placement of primer tRNA.
•
end also shows wild type
This suggests that the IN sequences
is dispensable for the genomic placement of primer tRNA.
SVC21 GR containing a
rnut~tion
Mutant virus
in the connection domain of RT, has been
shown to have non-selective viral tRNA packaging. and lacks mature Pol
186
•
gene sequences,
i.e.
p66 RT and p32
IN sequences.
Primer extension
analysis of SVC21 GR viral RNA shows a significant reduction of primer
tRNA Ly.3 placement cnte the HIV-I genome.
several possibilities:
genomic
plc!lcement.
1}
2}
This cou Id be explained by
Select primer tRNA may be a perquisite for
Genomic
placement
does
not
Pr160g·g-pol is not packaged into the viral particles.
oceur
because
3) The mutated
sequences in the SVC21 GR mutant(tryptophan repeat sequence) directly
blacks the mechanism of primer tRNA genornic placement in vivo .
•
187