Journal of General Virology (1993), 74, 2619-2628. Printed in Great Britain
2619
The cytopathic effect of human immunodeficiency virus is independent of
high levels of unintegrated viral DNA accumulated in response to
superinfection of cells
Anne G. Laurent-Crawford and Ara G. H o v a n e s s i a n *
Institut Pasteur, Unitd de Virologic et d'Immunologie Cellulaire (UA C N R S 1157), 28 rue du Dr Roux,
75724 Paris Cddex 15, France
Large quantities ofgenome-sized viral DNA are detected
in the nucleoplasm of CD4 ÷ T cells infected with human
immunodeficiency virus type 1 (HIV-1). This unintegrated HIV DNA is in the form of both circular and
linear species. Accumulation of such DNA occurs
gradually during a 5 day HIV infection and is correlated
with the proportion of cells involved in the production of
HIV proteins. To pinpoint the stage in a synchronized
HIV infection during which accumulation of HIV DNA
occurs, high titres of HIV were employed to infect CEM
cells to infect the majority of cells by the input virus. By
this latter infection, more than 95 % of cells became
producers of HIV proteins at 48 h post-infection (p.i.)
concomitantly with the development of the c.p.e, of
HIV, manifested by formation of syncytia and induction
of cell death by apoptosis. Addition of azidothymidine
(AZT) or neutralizing anti-gp 120 monoclonal antibodies
at 8 h p.i. did not alter the course of virus infection nor
the amount of virus produced at 48 h p.i. but the
accumulation of unintegrated HIV DNA was drastically
reduced. These results indicate that viral DNA accumulates as a result of superinfection of cells late in the virus
cycle. The development of the c.p.e, of HIV was
inhibited in the presence of neutralizing antibodies,
whereas in the presence of AZT the accumulation of
unintegrated HIV DNA was completely blocked without
apparent effect on the c.p.e. These observations indicate
that the c.p.e, of the HIV infection, which is manifested
by syncytium formation and apoptosis, does not require
superinfection of cells or accumulation of unintegrated
viral DNA.
Introduction
phages by binding to its principal receptor, the CD4
molecule (Marsh & Dalgleish, 1988). After viral entry,
HIV RNA is reverse-transcribed in the cytoplasm by the
viral transcriptase to produce the proviral DNA, which
is then integrated into the host chromosomal DNA (for
a recent review, see Cullen, 1991). In general, HIV
infection of cell cultures results in the generation of an
acute and/or chronic infection. In both cases, virus is
produced and becomes released by budding at the
cellular membrane. Acute infection is characterized by a
typical c.p.e, manifested by ballooning of cells, formation
of syncytia and subsequently by cell death. On the other
hand, chronically infected cells do not show this typical
c.p.e, despite their constant capacity to produce infectious HIV particles. This difference between acute and
chronic infection has been reported to be due in part to
a lower amount of virus particles produced and downregulation of CD4 receptors in persistently infected cells
(Stevenson et al., 1988). Recently, our group and others
have suggested that the c.p.e, of HIV-1 and HIV-2 is
associated with apoptosis in CD4 + cell cultures (LaurentCrawford et al., 1991 ; Terai et al., 1991). Apoptosis is a
Human immunodeficiency virus (HIV) is considered to
be the aetiological agent of AIDS in which CD4 + T
lymphocytes progressively become depleted (Rosenberg
& Fauci, 1991). The precise mechanisms responsible for
the depletion of CD4 + T lymphocytes is not yet clear.
Recently, programmed cell death or apoptosis has been
suggested to be responsible, at least in part, for T4
lymphocyte depletion in HIV-seropositive individuals
(Ameisen & Capron, 1991; Ameisen, 1992). Viral
infection may play a major role in the immunopathogenesis of HIV infection. In favour of this latter
explanation are a number of observations indicating that
disease progression is correlated with the emergence of
virus variants characterized by increasing cytopathic
phenotypes (Cheng-Mayer et al., 1988; Feny6 et al.,
1988; Tersmette et at., 1988; Schellekens et al., 1992) and
the close correlation between viral load, decreasing
CD4 + T cell count and disease progression (Schnittmann
et al., 1990; Hsia & Spector, 1991 ; Bagasra et al., 1992).
HIV infects lymphocytes, monocytes and macro0001-1745 © 1993 SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 06:56:46
2620
A. G. L a u r e n t - C r a w f o r d and A. G. Hovanessian
physiologicalcell suicide mechanism occurring in metabolically active cells and is manifested by the activation of
a Ca2÷-dependent nuclear DNase responsible for the
cleavage of chromatin at nucleosomal junctions (Duvall
& Wyllie, 1986). Accordingly, a classical DNA fragmentation was observed in CEM cells during acute
infection. In contrast, syncytium formation and DNA
fragmentation did not occur in chronically infected CEM
cells (Laurent-Crawford et al., 1991).
Previously, the c.p.e, of HIV in cell cultures has been
correlated with the accumulation of unintegrated viral
DNA occurring late in the course of infection (Shaw et
al., 1984; Muesing et al., 1985). This is analogous with
other retroviral systems, including avian viruses (Keshet
& Temin, 1979; Weller et al., 1980), feline leukaemia
viruses (Mullins et at., 1986), visna viruses (Haase, 1986)
and primate foamy viruses (Mergia & Luciw, 1991) in
which cell death has been associated with the accumulation of unintegrated retroviral DNA as a result of
massive second-round superinfection (Weller et al.,
1980). In the case of HIV infection, it has also been
observed that accumulation of unintegrated viral DNA
is due to superinfection (Robinson & Zinkus, 1990;
Pauza et al., 1990). These DNA species are in three major
forms: a linear copy of the viral genome with two long
terminal repeat (LTR) sequences and two circular DNA
species with one or two LTRs (Varmus & Swanstrom,
1984). Accumulation of unintegrated HIV DNA has also
been observed in vivo in lymph node tissues from AIDS
patients (Shaw et at., 1984). Moreover, owing to very
sensitive detection methods now available, it has been
shown that high levels of unintegrated HIV DNA can be
detected in the brain of AIDS dementia patients (Pang et
al., 1990) as well as in quiescent T lymphocytes from
asymptomatic individuals (Bukrinsky et al., 1991). A
recent study in macaques infected with simian immunodeficiency virus has indicated that much of the viral
DNA is unintegrated in most tissues taken at the time of
autopsy (Hirsch et al., 1991).
The role of unintegrated viral DNA in the evolution of
AIDS in HIV-infected patients remains to be investigated. On the other hand, by using a synchronous HIV
infection of cell cultures, we further confirm that the
accumulation of unintegrated viral DNA is due to
superinfection of cells at later stages of virus infection. In
addition, we demonstrate that this unintegrated HIV
DNA is not essential for the typically observed c.p.e, of
HIV.
Methods
H I V and cells. The HIV-1 Lai isolate previously referred to as HIV1 Bru (Wain-Hobson et al., 1991) was used in this study to infect CEM
clone 13 cells which are derived from the human lymphoblastoid cell
line CEM (ATCC-CCL 119) that expresses the T4 antigen to a high
level. Cells were cultured in suspension medium RPMI-1640 (GibcoBRL) containing 10% (v/v) fetal calf serum and 2 ~tg/ml polybrene
(Sigma).
Acute synchronous infection o f C E M cells. An HIV-1 stock with a
high infectivity was prepared by several consecutive passages of the
original HIV-1 Lai isolate on CEM cells (Laurent et al., 1990). The titre
of the viral stock was evaluated using CEM cells according to the
standard dilution procedure. In all infections, cells (5 × 106/ml) were
incubated with virus for 1 h at 37 °C before centrifugation and
resuspension in fresh culture medium at 1 × 106 cells/ml. Virus stocks
that gave a time course of infection lasting not more than 3 days were
considered appropriate for a synchronous infection of all ceils in the
culture, referred to as 1 synchronous infectious dose (containing 400 ng
of HIV particle-associated p25 per 106 cells). In such an infection, more
than 90 % of cells become producers of HIV proteins (according to an
immunoenzymatic assay; Laurent et al., 1989) at 48 h post-infection
(p.i.) at which time maximal c.p.e., manifested by ballooning of cells
and syncytium formation, is observed. Evidence that most of the cells
become infected by the input virus was provided by the observation
that infection is not modified in the presence of azidothymidine (AZT)
when added at 6 h p.i. In such a synchronous infection, cell lysis occurs
at day 3 p.i.
Monoclonal antibodies (MAbs). MAb N11/20 specific for gpl20 was
the generous gift of F. Nato of Hybridolaboratory in the Institut
Pasteur, Paris. This antibody recognizes an epitope in the V3 loop of
gpl20 (F. Traincard, unpublished observations). MAb 110/4 specific
to the V3 loop of gpl20 and MAb 25/3 specific to p25 were obtained
from Genetic Systems.
Preparation ofcell extracts. For the recovery of cytoplasm separately
from nucleoplasm, cells were disrupted in cytoplasm extraction buffer
containing 20 mM-Tri~HCI pH 7.6, 0.15 M-NaC1, 5 mM-MgCI2,
0-2 mM-PMSF, 100 units/ml aprotinin and 0.5% Triton X-100. After
centrifugation at 1000g, the supernatant contained the cytoplasm
whereas the intact nuclei were pelleted. The nuclei were then extracted
in lysis buffer containing 10 mM-Tris-HC1 pH 7-6, 0.4 M-NaC1, 1 mMEDTA, 0"2 mM-PMSF, 100 units/ml aprotinin (Iniprol) and 1% Triton
X-100. This nuclear extract was then centrifuged at 12 000 g to separate
the nucleoplasm from the high M r chromatin.
Analysis o f unintegrated H I V DNA. The nucleoplasm (prepared as
described above) contained the low M r DNA (cellular and unintegrated
HIV DNA) whereas the pellet of the nuclear extracts contained the
high M r chromatin. Nucleoplasm was incubated with 20 ~tg/ml RNase
for 1 h at room temperature, then 100 ~tg/ml proteinase K for 2 h at
37 °C, followed by extraction with phenol-chloroform-isoamylalcohol
(25:24: 1) and precipitation with ethanol. The pellet was analysed for
the presence of unintegrated HIV-1 DNA by slot blot and Southern
blot assays. The 3ZP-radiolabelled HIV DNA probe corresponding to
the entire HIV-1 genome was from p-Bru 2, a molecular clone of HIV1 Lai provided by K. Peden.
Analysis ofhistones. The c.p.e, of HIV-1 Lai manifested by syncytium
formation is associated with apoptosis (Laurent-Crawford et al., 1991).
Accordingly. the nucleoplasm of infected cells contains nucleosomes
which become accumulated as a consequence of chromatin fragmentation during apoptosis. The presence of nucleosomes could be
monitored either by extraction of the low M r DNA to analyse the
oligonucleosomal DNA fragments or by the recovery of histones
(H2A, H2B, H3 and H4) after solubilization in a non-ionic detergent,
such as SDS. For this latter purpose, crude nucleoplasm extracts were
diluted onefold in electrophoresis sample buffer (0.125M-Tris-HC1
pH 6-8, 2 % w/v SDS, 20 % glycerol and 1% 2-mercaptoethanol) and
boiled 5 min before analysis by polyacrylamide gel (15%) electrophoresis in SDS. Histones were clearly detectable by staining the gel
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 06:56:46
Cytopathic effect o f H I V infection
2621
(a)
i
i
i
i
i
I
(c)
(b)
lOO
1
2
kb
80
NC ~
23
1
60
"~
40
2
L ~
3 ~
C--~
4 ~
20
9.4
6.5
4.3
5~
2-3
I
0
1
2
3
4
5
6
Time p.i. (days)
Fig. 1. Accumulation of unintegrated HIV D N A in the nucleoplasm of CEM cells during multiple cycles of infection. CEM cells were
infected with HIV-1 at a dose corresponding to 20 ng of particle-associated p25 per 10n cells and the formation of syncytia was
monitored by light microscopy. The first syncytia appeared 2 days p.i. The maximal c.p.e, was observed at day 5 p.i. The experiment
observed was stopped 6 days p.i. since cell lysis was more than 70 %. (a) The proportion of cells expressing viral proteins. The kinetics
of infection was monitored by an immunoenzymaytic assay (Laurent et al., 1989) using MAbs 25/3 and 110/4 to reveal the major core
protein p25 and the external envelope glycoprotein gpl20, respectively. The ordinate gives the proportion (% positive cells) of cells
expressing p25 (O) and gpt20 (IlL (b) Slot blot assay. At different days p.i., cells were analysed for the accumulation of unintegrated
HIV-I DNA in the nucleoplasm (Methods). Material corresponding to 2 × l0 s cells was used at days 1 to 5 p.i. (c) Southern blot assay.
Unintegrated HIV DNA was extracted from the nucleoplasm of HIV-l-infected cells 5 days p.i. (Methods). DNA without (lane 1) or
with (lane 2) BamHI digestion was suspended in electrophoresis sample buffer (10 mM-Tris-HCl pH 8-0, 1 mM-EDTA) and analysed
by electrophoresis on a 1% agarose gel containing 0.5 lag/ml ethidium bromide at 80 V for 5 h. On the right is the position of DNA
markers. On the left NC, L and C indicate the position of the nicked circular, linear and circular forms of the HIV-1 DNA, respectively.
In sections (b) and (c), a z2P-labelled probe corresponding to the entire HIV-1 genome was used. Autoradiographs are shown.
with Coomassie blue (Laurent-Crawford et al.,
1991). Material
corresponding to 0"5 x l0 s cells was used.
Detection of viral antigens by immunoblot analysis and p25 capture
assay. Crude cytoplasmic extracts were diluted onefold in electrophoresis sample buffer and analysed by SDS PAGE. Cellular proteins
were stained with Coomassie blue; viral proteins were revealed by
immunoblot assays using HIV-l-positive sera and revealed by l~Ilabelled Protein A (Amersham; Laurent-Crawford et al., 1991).
Quantification of HIV-1 p25 in culture supernatants was performed by
using the p25 Core Profile ELISA (Du Pont).
Results
A multiple cycle kinetics in cells infected with H I V at
low multiplicity
All the experiments were carried out with a stock o f H I V 1 Lai ( W a i n - H o b s o n et al., 1991; previously referred to
as HIV-1 Bru) prepared on C E M cells (Laurent et al.,
1990). Routinely, after 1 h o f incubation, the input virus
was removed and cells were suspended in fresh culture
medium. A t a dose o f H I V corresponding to 20 ng o f
particle-associated p25 per 106 cells, the first signs o f
the c.p.e, were clearly observed 2 to 3 days p.i. by
vacuolization o f cells and appearance o f syncytia.
Fig. 1 gives a typical kinetics o f infection showing
the p r o p o r t i o n o f cells involved in the synthesis o f
viral proteins, and the accumulation o f unintegrated
HIV-1 D N A . M A b s specific for the HIV-1 envelope
glycoprotein g p l 2 0 and the m a j o r core protein p25 were
employed here in an i m m u n o e n z y m a t i c staining assay to
estimate the p r o p o r t i o n o f H I V - p r o d u c i n g cells. D u r i n g
the course o f infection, the p r o p o r t i o n o f cells producing
H I V proteins gradually increased reaching 82 and 95 %
for g p l 2 0 and p25, respectively, on day 5 p.i. The
difference between the detection o f g p l 2 0 and p25 is due
p r o b a b l y to the sensitivity o f the assay for each protein.
Consequently, the n u m b e r o f H I V - p r o d u c i n g cells at
5 days p.i. could be considered to be m o r e than 90 %,
during which time only a very small p r o p o r t i o n o f
infected cells remained as single cells. At this stage,
syncytium f o r m a t i o n was maximal and was associated
with apoptosis as we have reported previously (LaurentC r a w f o r d et al., 1991). Infected cells became lysed on day
6 p.i.
A c c u m u l a t i o n o f unintegrated HIV-1 D N A in the
infected cells was also gradual. I n fact very little H I V
D N A was observed in the nucleoplasm o f infected cells
1 to 2 days p.i. However, f r o m day 3 onwards there
was a dramatic increase which was highly correlated with
the p r o p o r t i o n o f H I V - p r o d u c i n g cells (Fig. 1). A
possible explanation for the gradual increase in the
p r o p o r t i o n o f H I V - p r o d u c i n g cells is the fact that only a
small n u m b e r o f cells become infected by the input virus.
Accordingly, only a very small p r o p o r t i o n o f cells were
f o u n d to be producers o f H I V proteins on day 1 p.i. (Fig.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 06:56:46
2622
A. G. Laurent-Crawford and A. G. Hovanessian
Table 1. A Z T added 6 h p.i. during multiple cycles of
H I V infection reduces the proportion of infected cells
and virus production*
Agent
Time of
addition (h)
Positive cells
(%) on day 4
Virus production
on day 5
[p25 (ng/ml)]
(% inhibition)
None
AZT
AZT
AZT
6
24
48
55
5
12
25
1158
176 (85)
258 (78)
548 (53)
* CEM cells were infected with HIV-1 as in the legend of Fig. 1. In
separate but similarly infected cultures, AZT (5 gM) was added at 6, 24
and 48 h p.i. On day 4, the proportion of HIV protein-expressing cells
was monitored by an immunoenzymatic assay using MAb 25/3 specific
for p25 (Laurent et al., 1989). Without AZT, the proportion of cells
positive for HIV proteins was 55 and 95% on days 4 and 5,
respectively. Viral production was measured on day 5 p.i. by assaying
the concentration (ng/ml) of particle-associated p25 in the culture
supernatant (percentage inhibition is given in parentheses).
(a)
1
(b)
2
3
gpl60 __
gpl20 - -
1
2
3
4
~
p68 - p55-
4
,~.....
until all cells become infected. This is p r o b a b l y the case,
since addition o f poly(A), poly(U), an inhibitor o f viral
entry ( L a u r e n t - C r a w f o r d et al., 1992a), at day 1 p.i.
blocked the second and successive r o u n d s o f infection
(data not shown). Similarly, addition o f A Z T at days 1
and 2 p.i. resulted in the inhibition o f viral p r o d u c t i o n
due to its inhibitory effect on the successive r o u n d s o f
infection (Table 1).
Southern blotting analysis o f the unintegrated H I V
D N A accumulated at day 5 p.i. revealed three m a j o r
forms migrating at positions corresponding to 6.0, 9-5
and approximately 25 kb (in some experiments migrating
at positions 20 to 23 kb) (Fig. 1). The 9"5 kb D N A b a n d
corresponds to the linear full-length genome o f HIV-1
whereas the 6.0 kb and the 25 kb bands should corres p o n d to supercoiled and nicked circular species with
one or two L T R s , in accord with previously published
observations (Charneau & Clavel, 1991; K i m et al.,
1989; Pauza et al., 1990). Digestion o f this HIV-1
g e n o m e with BamHI at the unique site at position 8520
( W a i n - H o b s o n et al., 1985) resulted in one b r o a d band,
consisting o f the circular forms converted into linear
forms o f 9 and 9"5 kb and the large fragment o f 8-6 kb
cleaved f r o m the linear species (Fig. 1).
Establishment of a synchronous infection of the majority
of cells in a culture
~....... ~ '
gp41 - p40 /
p25
Fig. 2. Synthesis of HIV-1 proteins during the course of a synchronous
infection of CEM cells. CEM cells were infected with 1 synchronous
infectious dose of HIV-1 (Methods: culture supernatant containing
400 ng of particle-associated p25 per 106 cells) and aliquots of such
infected cells (5 x 106) were analysed for the synthesis of viral proteins
by metabolic labelling with [asS]methionine at different times: 16 to
24 h (lanes 1), 24 to 32 h (lanes 2), 48 to 56 h (lanes 3) and 72 to 80 h
(lanes 4). Extracts from cells (a) and the virus pellets (b) were then
immunoprecipitated with an HIV-l-positive serum and analysed by
SDS-PAGE and fluorography. For metabolic labelling, cells were
incubated in MEM without L-methionine or serum, but supplemented
with 200gCi/ml of L-[~SS]methionine (specific activity> 1000
Ci/mmol) for 8 h for each point (Laurent et al., 1989). The position
of HIV-I proteins is indicated on the left: the precursor of the
envelope glycoprotein (gp160), the external (gp120) and transmembrane (gp41) envelope glycoproteins, the reverse transcriptase
(p68), the gag precursor (p55) and its partially cleaved product (p40),
and the major core protein (p25).
1). Virus p r o d u c e d f r o m the first r o u n d o f infection
might then infect uninfected cells. Thus, input infectious
virus can be amplified by successive rounds o f infections
The experiments described in Fig. 1 demonstrated that at
a dose of H I V corresponding to 20 ng o f p25 per 106
cells, only a small p r o p o r t i o n o f cells become infected by
the input virus. Thus investigating the relationship o f the
accumulation o f unintegrated H I V D N A to the c.p.e, o f
H I V required the use o f infection conditions in which a
majority o f cells become infected by the input virus. This
was achieved arbitrarily by the use o f higher doses o f
H I V that corresponded to 400 ng o f particle-associated
p25 per 106 cells. This dose was referred to as 1
synchronous infectious dose. At this dose the course o f
infection was 3 days. In such infections, synthesis o f
HIV-1 proteins was detectable as soon as 1 6 h p . i . ,
reaching a maximal synthesis by 48 h p.i. (Fig. 2) at a
time when m o r e than 90 % o f cells became producers o f
H I V proteins (detected by an i m m u n o e n z y m a t i c assay).
The c.p.e, o f the H I V infection which was manifested by
ballooning o f cells and syncytium f o r m a t i o n was observed at 48 h p.i. M o s t o f the unintegrated H I V D N A
became accumulated between 24 and 48 h p.i. (Fig. 3).
To determine the shortest time necessary for virus
adsorption, entry and reverse transcription o f H I V R N A
to initiate a s y n c h r o n o u s infection o f almost all cells in
the culture, A Z T was added at different times after virus
adsorption. As expected, addition o f A Z T with the virus
or up to 2 h after virus adsorption resulted in the
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 06:56:46
Cytopathic effect of H I V infection
C.p.e. -
-
-
+
(a)
-
(b)
Nll/20
1
p25-positive cells (%) 0
0
50
95
93
.
.
.
2
3
4
5
6
7
8
1
2
~
,~-
110/4
3
4
96
gpl20-
Anti-gp 120 M A b .
2623
=
~
+
p68 p55 gp41 -
Time p.i. (h)
0
8
24
48
48
48
p40 /
p25-
NC-
~
........
~
. . . . . . ,~.-
::~i~:;~°~
Fig. 4. C h a r a c t e r i z a t i o n o f the s y n c h r o n o u s HIV-1 infection o f C E M
cells. The effect o f A Z T a n d M A b s o n H I V p r o t e i n synthesis in C E M
cell cultures was studied d u r i n g the s y n c h r o n o u s infection as in Fig. 2
a n d 3. A Z T (5 gM) was a d d e d t o g e t h e r w i t h the virus [at 0 h; (a) lane
C-
Fig. 3. U n i n t e g r a t e d H I V D N A a c c u m u l a t e s as a c o n s e q u e n c e o f
superinfection o f cells d u r i n g a s y n c h r o n o u s infection of C E M cultures.
C E M cells were infected with 1 s y n c h r o n o u s infectious dose o f HIV-1
(as in the legend of Fig. 2) and analysed for the accumulation of
unintegrated HIV DNA at 8, 24 and 48 h p.i. (as in Fig. I c). An
autoradiograph is shown. The c.p.e, was monitored by the formation of
syncytia (indicated by plus symbol; minus symbol indicates no
syncytia). The proportion of cells producing HIV-1 p25 was estimated
using MAb 25/3 (as in Fig. la). MAbs 110/4 and Nll/20 (at
10 lag/ml; the last two lanes, respectively) were added to the infected
cultures at 8 h p.i. and the cultures were analysed at 48 h p.i. Both
antibodies blocked the formation of syncytia and the accumulation of
unintegrated HIV DNA but did not have any apparent effect on the
proportion of p25-positive cells.
inhibition o f viral protein p r o d u c t i o n assayed at 48 h p.i.
(Fig. 4a). The level o f viral protein p r o d u c t i o n started to
increase when A Z T was added 4 h p.i. (clearly d e m o n strated by the presence o f gpl20), 4 h being the shortest
delay required for synthesis of full-length linear viral
D N A f r o m the input viral R N A ( K i m et al., 1989).
However, A Z T added f r o m 6 h onwards did not cause
any significant modification o f viral p r o d u c t i o n (Fig. 4 a,
lanes 6 and 8). These results suggest that the majority o f
cells b e c o m e infected 6 to 8 h after the addition of virus,
and that this period is necessary for the reverse
transcription of the input viral genomic R N A into
proviral D N A . F u r t h e r m o r e these observations confirm
that under these experimental conditions cells were
synchronously infected by the input virus, therefore
generating an almost single cycle infection. This is in
contrast to the 5 to 6 day multiple cycles of infection
described in Fig. 1 since addition of A Z T , even at
2] or at 0.5, 1, 2, 4, 6 and 8 h after virus adsorption (a, lanes 3, 4, 5, 6,
7 and 8). Lane 1 represents the sample without AZT. MAbs Nll/20
and 110/4 against the V3 loop of gpl20 were added at 8 h p.i. and at
concentrations of 1 ~tg/ml (b, lanes 1 and 3) or 10 gg/ml (b, lanes 2 and
4). The different cultures were analysed by immunoblot analysis using
an HIV-l-positive serum (Methods) at 48 h p.i. Autoradiographs are
shown. On the left is shown the position of HIV-I proteins.
24 h p.i., resulted in a 78 % reduction o f virus p r o d u c t i o n
due to inhibition of infection of the remaining uninfected
cells (Table 1).
Higher doses o f H I V (corresponding to 400 ng of
p25/10" cells) were required here to achieve a synchronous infection of C E M cells, since a very small p r o p o r t i o n
of H I V particles become b o u n d to C E M cells. In fact less
than 2 % of H I V particles are found to bind to C E M cells
and, furthermore, only 50 % of the b o u n d virus enters
cells (Krust et al., 1993), implying that the infectivity of
the input virus is a r o u n d 1 % . Thus the virus stocks
contain a high p r o p o r t i o n of defective virus particles
p r o b a b l y due to partial but continual leakage of gp120
f r o m virus particles reducing their affinity to bind to
C D 4 receptor molecules.
The effect of anti-gpl20 neutralizing antibodies on the
synchronous infection
The synchronous H I V infection o f C E M cells was
characterized by M A b s specific for gpl20. M A b s 110/4
and N l l / 2 0 , b o t h recognizing the V3 loop on g p l 2 0 of
HIV-1 Lai, completely neutralized HIV-1 Lai infection
without significantly affecting the binding of gp 120 to the
C D 4 receptor ( K i n n e y - T h o m a s et al., 1988; Linsley
et al., 1988). U n d e r the synchronous infection conditions, addition o f either a n t i b o d y together with H I V
resulted in the inhibition of virus infection as expected.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 06:56:46
2624
A. G. Laurent-CrawJord and A. G. Hovanessian
However addition of these antibodies at 8 h p.i. did
not modify the course of infection nor did it affect the
intensity of viral production. Fig. 4 (b) shows the pattern
and the quantity of HIV proteins found in cells at
48 h p.i. It should be noted that both antibodies were
functional since they blocked the syncytium formation as
well as ballooning of cells routinely observed at 48 h p.i.
in cultures without the antibodies (Fig. 5). Furthermore,
these antibodies almost completely blocked the accumulation of unintegrated HIV DNA (Fig. 3, lanes
labelled + for MAb anti-gpl20). Therefore in this
synchronous infection, accumulation of the unintegrated
HIV DNA results from superinfection, as previously
reported by others using longer times of infection
(Robinson & Zinkus, 1990; Pauza et al., 1990; Besansky
et al., 1991). Our results also indicate that superinfection
is not necessary for optimal viral protein expression
when the majority of cells become infected by the input
virus.
A Z T added at 6 h p.i. blocks the accumulation of
unintegrated viral DNA without modifying the c.p.e, of
HIV
Fig. 5. Inhibition of the c.p.e, of HIV-1 by anti-gpl20 antibody but not
by A Z T treatment. C E M cells under synchronous infection conditions
(Fig. 2 to 4) were incubated with M A b 110/4 (10 gg/ml) or A Z T
(5 pM). Both agents were addd at 8 h p.i. The c.p.e, was monitored
under a light microscope by the formation of syncytia. (a) Uninfected
CEM cells. (b) HIV-infected CEM cells. (c) HIV-infected cells plus
anti-gpl20 at 8 h p.i. (d) HIV-infected cells plus A Z T at 8 h p.i.
During a synchronous infection, HIV RNA replication
was blocked by the addition of AZT at different times
following virus adsorption. At 48 h, virus infection was
monitored by the development of c.p.e. (formation of
syncytia), the accumulation of unintegrated HIV DNA
and by the production of virus, estimated by the
concentration of p25 in the culture medium (Fig. 6). In
a previous study, we reported that the c.p.e, ofHIV-1 Lai
and HIV-2 ROD is associated with apoptosis in CD4 T
cell cultures (Laurent-Crawford et al., 1991) and that this
effect is mediated by the expression of HIV envelope
glycoproteins (Laurent-Crawford et al., 1992b). One
marker for apoptosis or programmed cell death is the
presence of cellular low M r DNA consisting of multimers
of a nucleosome-length unit of 200 bp. As an alternative
to this method, we have demonstrated that Coomassie
blue staining of histones associated with these low Mr
DNA fragments can be used as a convenient assay to
monitor apoptosis in HIV-infected cells (LaurentCrawford et al., 1991). Accordingly, the detection of
histones was used to quantify the relative level of
apoptosis occurring in these infected cultures (Fig. 6).
Addition of AZT together with the virus, or 1 h after
virus adsorption, resulted in a dramatic inhibition of
virus production, development of syncytia and apoptosis.
On the other hand, AZT added from 6 h onwards did not
cause any apparent modification in the kinetics and the
production of virus nor in the development of syncytia
and apoptosis (Fig. 5 and 6). Treatment with AZT was
effective, since accumulation of the unintegrated HIV
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 06:56:46
Cytopathic effect of H I V infection
Table 2. Single cell killing by lysis in the presence of
anti- V3 MAbs*
00
8O
Virus
production
[~ Syncytia
(ng p25/ml)
m
60
~p25
40
~i~ Apoptosis
0
2625
~)
~)~
I|)I!I
Jb,l)ml,
,lil,
l
0
1
4
6
24
Time of AZT treatment (h)
-
Low Mr DNA
0
1
4
6
24
Synchronous infection Day 2
Cell lysis
Apoptosis
Day 3
Day 2
Day 3 Day 4
No addition
MAb 110/4 at 8 h
AZT at 8 h
766
821
1173
1255
1306
1488
+ +
+ +
+
+
+ +
+
+ +
* CEM cells were infected with one synchronous infectious dose of
HIV-1 (as in the legend of Fig. 2 to 4) and analysed for the production
of virus on days 2 and 3 p.i., apoptosis on day 2 p.i. and cell lysis on
days 3 and 4 p.i. Virus production was measured by the concentration
of p25 in the culture supernatant. Apoptosis was monitored by the
presence of histones as in the legend of Fig. 6. Cell lysis was monitored
by the trypan blue inclusion assay. MAb 110/4 against the V3 loop
(10 gg/ml) and AZT (5 gM) were added 8 h p.i.
I
Fig. 6. AZT added at 6 h p.i. blocks accumulation of unintegrated viral
DNA but does not modify the c.p.e, of HIV infection. CEM cells were
infected with HIV-I as in Fig. 2 but AZT (5 gM) was added at different
times during infection: with the virus (0 h) or 1, 4, 6 and 24 h p.i. At
48 h p.i., all cultures were examined under a light microscope for the
development of the c.p.e, and the number of syncytia was estimated
(column syncytia). The production of virus (column p25) was estimated
by measuring the amount of p25 in culture media; the value in the
culture without AZT corresponded to 156 gg/ml. Apoptosis was
monitored by the presence of histones (H2A, H2B, H3 and H4), the
amount of which was estimated by scanning the stained gels (Methods;
Laurent-Crawford et al., 1991). The insert below shows the unintegrated HIV DNA accumulated in the nuclei of infected cells untreated
(lane - ) or treated with AZT at different times (h, shown above lanes).
The autoradiograph shows the results of a slot blot assay. The probe
was the entire HIV-1 genome. An autoradiograph is shown.
DNA was almost completely inhibited (Fig. 6, insert).
These results confirm once again that an interval of a
minimum of 6 h was required during which time the
input viral RNA was reverse-transcribed into proviral
DNA to initiate the synchronous infection. The fact that
AZT added at 6 h p.i. did not modify the development of
the c.p.e, but drastically inhibited the accumulation of
unintegrated HIV DNA suggests that these two events
are not related.
Single-cell killing by lysis in the presence of anti-V3
MAbs
Under well defined experimental conditions, the c.p.e, of
HIV-1 and HIV-2 in CD4 + T lymphocytes is tightly
associated with apoptosis (Terai et al., 1991; LaurentCrawford et al., 1991, 1992b), in the course of which
cellular DNA becomes fragmented at internucleosomal
junctions. We have recently demonstrated that apoptosis
is triggered by cell membrane expression of the mature
HIV envelope glycoproteins, the gpl20/gp41 complex,
and their interaction with CD4 receptor molecules
(Laurent-Crawford et al., 1992b, 1993). In the case of the
HIV Lai isolate, syncytium formation and apoptosis
induction were closely associated as both events require
functional envelope glycoproteins and CD4 receptors.
Despite this, we have been able to dissociate syncytium
formation from apoptosis. For example, MAb OKT4
against the CD4 molecules inhibited apoptosis without
affecting syncytium formation, whereas another antibody specific for the transmembrane envelope glycoprotein gp41 blocked syncytium formation without
affecting the induction of apoptosis (Laurent-Crawford
et al., 1993). Taken together, these observations indicate
that apoptosis can occur in HIV-infected cells irrespective of syncytium formation. In accord with this,
infection of CEM cells with both syncytium-inducing
(HIV-2 ROD) or non-syncytium-inducing (HIV-2
EHO) cytopathic HIV-2 strains resulted in apoptosis
(Rey et al., 1989a, b, unpublished results).
Apoptosis could be used as an early signal for the
prediction of the c.p.e, during the HIV infection (Table
2). For example, in the synchronous type of HIV
infection, apoptosis is observed at day 2 p.i., at a time of
maximal production of HIV, i.e. as measured by the
maximal level of viral protein synthesis. At day 2 p.i., no
cell lysis occurs which is shown by the trypan blue
exclusion assay. Eventually apoptotic cells disintegrate
owing to cell lysis by day 4 p.i. (Table 2). Addition of
AZT at 8 h p.i. blocks accumulation of the unintegrated
HIV DNA due to reinfection of cells (Fig. 6) but does not
affect the course of infection nor the induction of
apoptosis (Table 2). On the other hand, addition of MAb
specific for the V3 loop at 8 h p.i. completely blocks
syncytium formation and induction of apoptosis. Cells in
this latter culture will eventually die owing to cell lysis by
day 5 p.i. (Table 2). Several reasons may account for lysis
of single cells in the culture with the anti-V3 MAb. For
example, the continual expression of high levels of viral
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 06:56:46
2626
A. G. L a u r e n t - C r a w f o r d and A. G. Hovanessian
RNA and protein synthesis (Somasundaran & Robinson,
1988) combined with the dramatic inhibition of cellular
protein synthesis (Agy et al., 1990) both occurring during
the late stages of HIV infection, may lead to severe shutoff of cellular metabolism resulting in cell lysis. Thus, the
HIV-mediated c.p.e, in cell cultures, whether associated
or not with apoptosis, will eventually terminate in cell
lysis.
Discussion
Analogous to the c.p.e, of some retroviruses (Keshet &
Temin, 1979; Wetler et al., 1980; Mullins et al., 1986;
Haase, 1986; Mergia & Luciw, 1991), the c.p.e, of HIV
has been correlated with the accumulation of unintegrated viral DNA late in the course of virus infection
(Shaw et al., 1984; Muesing et al., 1985; Pauza & Singh,
1990). The results presented here, however, rule out any
correlation between the accumulation of unintegrated
viral DNA and the c.p.e, of HIV. In a synchronous
infection of CEM cultures, addition of AZT 6 h p.i.
blocked the accumulation of the unintegrated DNA
without any apparent effect on the c.p.e, or on the
amount of virus produced (Fig. 3, 4 and 5). The
accumulation of unintegrated HIV DNA is probably due
to superinfection of cells as reported previously (Pauza &
Singh, 1990; Robinson & Zinkus, 1990). In agreement
with this, addition of MAbs against gpl20 (which
neutralize HIV infection) 6 h p.i. blocked accumulation
of unintegrated HIV DNA without affecting the kinetics
nor the amount of HIV particles produced (Fig. 2 and 3).
These MAbs therefore blocked accumulation of unintegrated HIV DNA by inhibiting superinfection of previously infected cells.
The lack of correlation between the c.p.e, and
accumulation of unintegrated HIV DNA was clearly
demonstrated using this synchronous infection in which
almost all cells became infected by the input virus. Under
these experimental infection conditions, the c.p.e, was
at its maximum at 48 h p.i., during which time more
than 90% of cells were found to be producers of HIV
proteins. Addition of AZT at 6 h p.i. did not modify the
production of virus or the development of the c.p.e. In
these experiments, the c.p.e, of HIV was monitored by
the ballooning of cells and syncytium formation, and
also by the presence of histones associated with low M r
DNA that accumulate in the nuclei of infected cells as a
consequence of apoptosis (Laurent-Crawford et al.,
1991). Apoptosis, which should be differentiated from
uncontrolled cell death or cell lysis, is a physiological
event triggering cell death by the activation of a Ca 2+dependent nuclear DNase that cleaves the chromatin at
nucleosomal junctions (Duvall & Wyllie, 1986). This
characteristic fragmentation of the chromatin occurs in
metabolically active cells and can be used as a convenient
marker for cells that are condemned to die. Consequently, as apoptosis is assayed in the nuclei of intact
cells, it provides an early signal of events that subsequently will lead to inhibition of cellular functions.
In a synchronous infection, the various stages of HIV
replication could be investigated independently of each
other. It is important to note that most previously
published investigations have been based on HIV
infections which last at least 5 to 6 days, i.e. under
multiple cycles of infection as shown in Fig. 1. During
multiple cycles of infection, only a small proportion of
cells become infected by the input virus. For this reason,
the course of infection lasts several days to allow time for
amplification of the input virus and multiple rounds of
infection. Experimental HIV infections of the multiplecycle type are useful for comparing viral stocks but are
not quite adequate in studies dealing with specific phases
of HIV infection, such as accumulation of unintegrated
viral DNA and the c.p.e, including cell killing. For
example, a recent study which dissociated the accumulation of unintegrated viral DNA and cell killing during
HIV infection was based on an infection lasting more
than 5 days (Bergeron & Sodroski, 1992). Interestingly,
in this latter work the authors mention that addition of
AZT early during infection (up to 3 days p.i.) results in
a significant reduction in viral protein expression, thus
indicating that they are dealing with an infection of the
multiple-cycle type. One of the major drawbacks of a
multiple-cycle infection developing gradually in the cell
culture is that a real event triggered by HIV infection in
a small proportion of cells will not be detectable because
of dilution in the larger proportion of cells that are not
yet infected. Late in a multiple-cycle infection, due to a
severe down-regulation of CD4 molecules, the typical
c.p.e, of HIV does not occur and instead, cells start to die
by lysis. This is most probably due to severe inhibition of
cellular metabolism owing to high levels of viral RNA
and protein synthesis (Somasundaran & Robinson, 1988)
and also to a dramatic shut-off of cellular protein
synthesis (Agy et al., 1990).
Unintegrated HIV DNA becomes accumulated during
an acute infection as a result of superinfection of cells
(Robinson & Zinkus, 1990; Pauza et al., 1990). Unintegrated HIV DNA is also detectable in peripheral blood
mononuclear cells of AIDS patients not undergoing
therapy. However, after a few months of anti-retroviral
therapy the levels of such unintegrated DNA are
significantly reduced (Dickover et al., 1992). In cell
cultures, the unintegrated HIV DNA species are in three
major forms : a linear copy of the viral genome with two
LTRs and two circular DNA species with one or two
LTRs (Kim et al., 1989; Charneau & Clavel, 1991). The
precise role of the circular DNA species in the virus
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 06:56:46
Cytopathic effect of H I V infection
replication cycle is not clear. On the other hand, it has
been suggested that the linear form of DNA is the
immediate precursor for integration into the host
chromosome (Grandgenett & Mumm, 1990; Charneau
& Clavel, 1991). Accumulation of unintegrated HIV
DNA does not occur in chronically infected T cells since
because of down-regulation of CD4 molecules they
cannot be superinfected (Stevenson et al., 1988; LaurentCrawford et al., 1991). Interestingly, accumulation of
unusual, high M r extrachromosomal forms of HIV DNA
has been observed in persistently HIV-infected monocytic cells (Pauza & Galindo, 1989; Pauza & Singh,
1990). Here we have demonstrated that accumulation of
unintegrated HIV DNA is not correlated with the c.p.e.
of HIV. Such a correlation has already been questioned
in the visna virus model, with the suggestion that the
envelope glycoprotein, capable of fusing from within and
from outside, is a more likely candidate (Haase, 1986).
This hypothesis is in agreement with data obtained in the
HIV model in which the expression of viral envelope
glycoproteins has been closely correlated with the c.p.e.
(Sodroski et al., 1986; Lifson et al., 1986; LaurentCrawford et al., 1992b, 1993).
This work was supported by a grant from Association Nationale de
la Recherche sur le SIDA in France.
References
AGY, M.B., WAMBACH, M., FOY, K. & KATZE, M.G. (1990).
Expression of cellular genes in CD4 positive lymphoid cells infected
by the human immunodeficiency virus, HIV-1 : evidence for a host
protein synthesis shut-off induced by cellular mRNA degradation.
Virology 177, 251-258.
AMEISEN, J.C. (1992). Programmed cell death and AIDS: from
hypothesis to experiment. Immunology Today 13, 388 391.
AMEISEN,J. C. & (;APRON,A. (1991). Cell dysfunction and depletion in
AIDS: the programmed cell death hypothesis. Immunology Today
12, 10~105.
BAGASRA,O., HAUPTMAN,S. P., HAROLD,D. O., L1SCHNER,W., SACHS,
M. & POMERANTZ, R.J. (1992). Detection of human immunodeficiency virus type 1 provirus in mononuclear cells by in situ
polymerase chain reaction. New England Journal of Medicine 326,
1385-1391.
BERGERON,L. & SODROSKI,J. (1992). Dissociation of unintegrated viral
DNA accumulation from single cell lysis induced by human
immunodeficiency virus type 1. Journal of Virology 66, 5777-5787.
BESANSKY, N.J., BUTERA, S.T., SINHA, S. & FOLKS, T.M. (1991).
Unintegrated human immunodeficiency virus type 1 DNA in
chronically infected cell lines is not correlated with surface CD4
expression. Journal of Virology 65, 2695-2698.
BUKRINSKY,M. I., STANWICK,T. L., DEMPSEY,M. P. & STEVENSON,M.
(1991). Quiescent T lymphocytes as an inducible virus reservoir in
HIV-1 infection. Science 254, 423-427.
CHARNEAU~P. & CLAVEL,F. (1991). A single-stranded gap in human
immunodeficiency virus unintegrated linear DNA defined by a
central copy of the polypurine tract. Journal of Virology 65,
2415 2421.
CHENG-MAYER, C., SETO, D., TATENO, D. & LEVY, J.A. (1988).
Biologic features of HIV-1 that correlate with virulence in the host.
Science 240, 81~82.
CULLEN, B. R. (1991). Regulation of human immunodeficiency virus
replication. Annual Review of Microbiology 45, 19-50.
2627
DICKOVER, R.E., DONOVAN, R.M., GOLDSTEIN, E., COHEN, S.H.,
BOLTON, V., HUTH, R.G., LIU, G. & CARLSON, J.R. (1992).
Decreases in unintegrated HIV DNA are associated with antiretroviral therapy in AIDS patients. Journal of AIDS 5, 31-36.
DUVALL, E. & WYLLIE, A. H. (1986). Death and the cell. Immunology
Today 7, 115-119.
FENYO, E.M., MORFELDT-MANSON,L., CHIODI, F., LIND, B., VON
GEGERFELT, A., ALBERT, J., ALBERT, E., OLAUSSON,E. & AS~O, B.
(1988). Distinct replicative and cytopathic characteristics of human
immunodeficiency virus isolates. Journal of Virology 62, 44144419.
GRANDGENETT, D.P. & MUMM, S. R. (1990). Unraveling retrovirus
integration. Cell 60, 3-4.
HAASE, A.T. (1986). Pathogenesis of lentivirus infections. Nature,
London 322, 130-136.
HIRSCH, V. M., ZACK,P. M., VOGEL,A. P. & JOHNSON,P. R. (1991).
Simian immunodeficiency virus infection of macaques: end-stage
disease is characterized by widespread distribution of proviral DNA
in tissues. Journal of AIDS 163, 976-988.
HSIA, K. & SPECTOR, S.A. (1991). Human immunodeficiency virus
DNA is present in a high percentage of CD4 ÷ lymphocytes of
seropositive individuals. Journal of AIDS 164, 47(~475.
KESHET, E. & TEMIN, H. (1979). Cell killing by spleen necrosis virus is
correlated with a transient accumulation of spleen necrosis virus
DNA. Journal of Virology 31, 376-388.
KIM, S., BYRN, R., GROOPMAN,J. & BALTIMORE,D. (1989). Temporal
aspects of DNA and RNA synthesis during human immunodeficiency virus infection: evidence for differential gene expression.
Journal of Virology 63, 3708 3713.
KINNEY-THOMAS, E., WEBER, J. N., McCLURE, J., CLAPHAM, P. R.,
SINGHAL, M. C., SHRIVER,M. K. & WEISS, R. (1988). Neutralising
monoclonal antibodies to the AIDS virus. AIDS 2, 25-29.
KRUST, B., CALLEBAUT,C. & HOVANESSIAN,A. G. (1993). Inhibition of
entry of HIV particles in cells by poly(A), poly(U). AIDS Research
and Human Retroviruses (in press).
LAURENT, A.G., KRUST, B., REY, M.A., MONTAGNIER, L. &
HOVANESSIAN,A. G. (1989). Cell surface expression of several species
of human immunodeficiency virus type 1 major core protein. Journal
of Virology 63, 4074-4078.
LAURENT, A.G., HOVANESSIAN, A.G., RIVIERE, Y., KRUST, B.,
REGNAULT,A., MONTAGNIER,L., FINDELI,A., KIENY,M. P. & GuY,
B. (1990). Production of a non-functional nef protein in human
immunodeficiency virus type 1-infected CEM cells. Journal of General
Virology 71, 2273 2281.
LAURENT-CRAWFORD,A. G., KRUST,B., MULLER,S., RIVIi~RE,Y., REYCUILLI~, M.A., Bt~CHET,J.M., MONTAGNIER,L. & HOVANESSIAN,
A.G. (1991). The cytopathic effect of HIV is associated with
apoptosis. Virology 185, 82%839.
LAURENT-CRAWFORD,A. G., KRUST, B., DESCHAMPSDE PAILLETTE,E.,
MONTAGNIER,L. & HOVANESSIAN,A. G. (1992a). Antiviral action of
polyadenylic-polyuridylic acid against HIV in cell cultures. AIDS
Research and Human Retroviruses 8, 285 290.
LAURENT-CRAWEORD,A.G., KRUST, B., REY, M.A., COINTE, D.,
COCCIA,E., RIVIi~RE,Y., MULLER,S., KIENY,M. P., DAUGUET,C. &
HOVANESSIAN,A. G. (1992b). Membrane expression of HIV envelope
glycoproteins triggers apoptosis in CD4 cells: a direct mechanism for
T4 cells depletion mediated by virus replication. In Retroviruses of
Human AIDS and Related Animal Diseases, pp. 35-41. Edited by
M. Girard & L. Valette. France: Pasteur M6rieux.
LAURENT-CRAWFORD,A. G., KRUST, B., RIVI[3RE,Y., DESGRANGES,C.,
MULLER, S., KIENY, M.P., DAUGUET,C. & HOVANESSIAN,A.G.
(1993). Membrane expression of HIV envelope glycoproteins triggers
apoptosis in CD4 cells. AIDS Research and Human Retroviruses 9,
761 773.
LIFSON, J. O., FEINBERG,M. B., REYES,G. R., RABIN, L., BONAPOUR,
B., CHAKRABARTI,S., MOSS, B., WONG-STAAL,F., STEIMER,K. S. &
ENGLEMAN,E. G. (1986). Induction of CD4-dependent cell fusion by
the HTLV-III/LAV envelope glycoprotein. Nature, London 323,
725 728.
LINSLEY, P. S., LEDBETTER,J.A., KINNEY-THOMAS,E. & Hu, S.L.
(1988). Effects of anti-gpl20 monoclonal antibodies on CD4 receptor
binding by the env protein of human immunodeficiency virus type 1.
Journal of Virology 62, 3695 3702.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 06:56:46
2628
A. G. Laurent-Crawford and A. G. Hovanessian
MARSH, M. & DALGLEISH,A. (1988). How do human immunodeficiency
viruses enter cells? Immunology Today 8, 369-371.
MERCIA, A. & LucIw, P.A. (1991). Replication and regulation of
primate foamy viruses. Virology 184, 475-482.
MUESING, M.A., SMITH, D. H., CABRADILLA,C. D., BENTON, C.V.,
LASKY, L.A. & CAPON, D.J. (1985). Nucleic acid structure and
expression of the human AIDS/lymphadenopathy retrovirus.
Nature, London 313, 450-458.
MULLINS, J. I., CHEN, C. S. 81; HOOVER, E. A. (1986). Disease-specific
and tissue-specific production of unintegrated feline leukemia virus
variant DNA in feline AIDS. Nature, London 319, 333 336.
PANG, S., KOYANAGI,Y., MILES, S., WILEY, C., VINTERS, H. V. & CHEN,
I. S. Y. (1990). High levels of unintegrated HIV-1 DNA in brain
tissue of AIDS dementia patients. Nature, London 343, 85-89.
PAUZA, C. D. & GAUNDO, J. E. (1989). Persistent human immunodeficiency virus type 1 infection of monoblastoid cells leads to
accumulation of self-integrated viral DNA and to production of
defective virions. Journal of Virology 63, 3700-3707.
PAUZA, C. D. & SINGH, M. K. (1990). Extrachromosomal HIV-1 DNA
in persistently infected U 937 cells. AIDS Research and Human
Retroviruses 6, 1027-1030.
PAUZA, C. D., GALINDO, J. E. & RICHMAN, D. D. (1990). Reinfection
results in accumulation of unintegrated viral DNA in cytopathic and
persistent human immunodeficiency virus type 1 infection of CEM
cells. Journal of Experimental Medicine 172, 1035-1042.
REY, M.A., KRUST, B., LAURENT, A.G., MONTAGNIER, L. &
HOVANESSlAN, A. G. (1989a). Characterization of HIV-2 envelope
glycoproteins: dimerization of the glycoprotein precursor during its
processing. Journal of Virology 63, 647 658.
REY, M. A., KRUST, B., LAURENT, A. G., GUIITARD, D., MONTAGNIER,
L. & HOVANESSIAN, A. G. (1989b). Characterization of an HIV-2
related virus with a smaller sized extracellular envelope glycoprotein.
Virology 173, 258 267.
ROBINSON, H. L. 8l; ZINKUS, D. M. (1990). Accumulation of human
immunodeficiency virus type 1 DNA in T cells: result of multiple
infection events. Journal of Virology 64, 4836-4851.
ROSENBERG, Z. F. & FAUCl, A. S. (1991). Immunopathogenesis of HIV
infection. FASEB Journal 5, 2382 2390.
SCHELLEKENS,P. TH. A., TERSMETTE,M., ROOs, M. TH. U, KEET, R. P.,
DEWOLF, F., COUTINHO,R. A. & MIEDEMA,F. (1992). Biphasic rate
of CD4 + cell count decline during progression to AIDS correlates
with HIV-1 phenotype. AIDS 6, 665 669.
SCHNITTMANN, S.M., GREENHOUSE, J.J., PSALLIDOPOULOS, M.C.,
BASELER, M., SALZMAN, N. P., FAUCI, A. S. & LANE, H. C. (1990).
Increasing viral burden in CD4* T cells in patients with human
immunodeficiency virus (HIV) infection reflects rapidly progressive
immunosuppression and clinical disease. Annals of Internal Medicine
113, 438-443.
SHAW, G. M., HAHN, B. H., ARYA, S. K., GROOPMAN, J. E., GALLO,
R.C. & WONG-STAAL, F. (1984). Molecular characterization of
human T-cell leukemia (lymphotropic) virus type III in the acquired
immune deficiency syndrome. Science 226, 1165 1171.
SODROSKI, J. G., GOH, W. C., ROSEN, A., CAMPBELL, K. & HASELTINE,
W.A. (1986). Role of the HTLV/LAV envelope in syncytia
formation and cytopathicity. Nature, London 322, 470-474.
SOMASUNDARAN, M. & ROBINSON, H.L. (1988). Unexpectedly high
levels of HIV-I RNA and protein synthesis in a cytocidal infection.
Science 242, 1554-1557.
STEVENSON,M., MEIER, C., MANN, A. M., CHAPMAN,N. & WASIAK,A.
(1988). Envelope glycoprotein of HIV induces interference and
cytolysis resistance in CD4 + ceils: mechanism for persistence in
AIDS. Cell 53, 483-496.
TERAI, C., KORNBLUTH, R.S., PAUZA, C.D., RICHMAN, D.D. &
CARSON, D. A. (1991). Apoptosis as a mechanism of cell death in
cultured T lymphoblasts acutely infected with HIV-1. Journal of
Clinical Investigation 87, 1710-1715.
TERSMETTE, M., DE GOEDE, R. E. Y., AL, B. J. M., WINKEL, I.N.,
GRUTERS, R. A., CUYPERS, H. T., HUISMAN, H. G. & MIEDEMA, F.
(1988). Differential syncytium-inducing capacity of human immunodeficiency virus isolates: frequent detection of syncytium inducing
isolates in patients with acquired immunodeficiency syndrome
(AIDS) and AIDS-related complex. Journal of Virology 62,
2026-2032.
VARMUS, H. & SWANSTROM, R. (1984). Replication of retroviruses. In
RNA Tumor Viruses, pp. 369-512. Edited by R. Weiss, N. Teich, H.
Varmus & J. Coffin. New York: Cold Spring Harbor Laboratory.
WAIN-HOBSON, S., SONIGO, P., DANOS, O., COLE, S. & ALIZON, M.
(1985). Nucleotide sequence of the AIDS virus, LAV. Cell 40, %17.
WAIN-HOBSON, S., VARTANIAN, J.P., HENRY, M., CHENCINER, N.,
CHEYNIER, R., DELASSUS, S., MARTINS, L. P., SALA, M., NUGEYRE,
M.T., GUETARD, D., KEATZMANN, D., GLUCKMAN, J.C.,
RISENBAUM,W., BARRE-SINOUSSI,F. & MONTAGNIER,L. (1991). LAV
revisited: origins of the early HIV-I isolates from Institut Pasteur.
Science 252, 961 965.
WELLER,S. K., JOY, A. E. & TEMIN,H. M. (1980). Correlation between
cell killing and massive second-round superinfection by members of
some subgroups of avian leukosis virus. Journal of Virology 33,
494-506.
(Received 6 April 1993; Accepted 27 July 1993)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 06:56:46