Journal of General Virology (1995), 76, 951-956. Printed in Great Britain
951
Processing of the plum pox virus polyprotein at the P3 6K1 junction is not
required for virus viability
Jos6 Luis Riechmann,'~ M a r i a Teresa Cervera and Juan Antonio Garcia*
Centro de Biologfa Molecular (CSIC-UAM) and Centro Nacional de Biotecnologfa, Campus Universidad Aut6noma,
28049 Madrid, Spain
Proteolytic processing of the potyvirus polyprotein is
mainly performed by the virus-encoded NIa protease,
whose cleavage sites are characterized by conserved
heptapeptide sequences. Partial processing at the cleavage site present between the P3 and 6K~ cistrons by the
plum pox potyvirus (PPV) NIa protease has been
previously shown to occur in vitro. We have now studied
the role ofpolyprotein processing at the P3-6K1 junction
in vivo, using a full-length PPV eDNA clone. PPV
mutant transcripts containing a histidine for glutamine
substitution in the cleavage site sequence (a change that
abolishes in vitro processability) are able to infect
Nieotiana clevelandii plants, indicating that normal
processing at the P3-6K 1 junction is not required for
virus viability. However, disease symptoms were not
detected and virus accumulation occurred after a second
site mutation was introduced into the 6K 1 cistron during
replication. This additional change did not restore the in
vitro processability of the mutant heptapeptide. Changes
at other positions in the heptapeptide (that only slightly
altered the in vitro processability of this NIa site) were
also engineered and it was found that these mutations
affected the time course and severity of the symptom
induction process. A possible regulatory effect on the
function of the potyvirus P3 + 6K 1 protein by processing
at the P3-6Kt junction is discussed in light of our
present results with PPV.
The potyvirus genome, a 10 kb RNA molecule, is
expressed through its translation into a unique polyprotein that undergoes extensive proteolytic processing
(Riechmann et al., 1992). The virus-encoded NIa
protease catalyses most of the proteolytic events,
processing the central and C-terminal regions of the
polyprotein (Fig. 1). NIa cleavage sites are defined by
conserved heptapeptides (amino acids - 6 to + 1, the
scissile bond located between - 1 and + 1 ; Dougherty et
al., 1988, 1989; Carrington & Dougherty, 1988; Garcia
et al., 1989b) and seven of these sequences (named A to
F and v; Fig. 1) are found along the potyvirus
polyprotein. That these function as NIa cleavage sites
has been demonstrated experimentally by N-terminal
sequencing of proteins purified from infected tissue (sites
B, D, E and F) and by analysis of the products of
proteolytic processing (from all seven sites) both in vitro
and in Escherichia coil. It is now known that each site has
different cleavage peculiarities (cis or trans processing,
partial or complete cleavage and different reaction
profiles; for review see Riechmann et al., 1992;
Dougherty et al., 1990). Nevertheless, there is still
controversy over whether the A site is actually used in
vivo and, in fact, the nature of the A heptapeptide as an
NIa cleavage site has often been overlooked. This
heptapeptide is present at similar positions in the
polyproteins of all 12 potyviruses sequenced to date
(Lain et al., 1989; see below) and it has been shown in
cell-free proteolytic processing experiments that the plum
pox virus (PPV) site A is partially cleaved in vitro (Garcfa
et al., 1992). In addition, the presence in plants infected
by tobacco vein mottling virus of two proteins of 42 and
37 kDa that immunoreact with antibodies raised against
a bacterially expressed 42 kDa protein corresponding to
the P 3 + 6 K 1 cistron (Fig. 1) supports the existence of
cleavage site A and suggests that, in vivo, it might also be
only partially processed (Rodriguez-Cerezo & Shaw,
1991). Therefore, three different gene products would
originate from this region of the potyvirus polyprotein:
P3, 6K1 and P3 + 6 K a. However, in vitro processing at
tobacco etch virus (TEV) site A has also been assayed
and, in this case, no cleavage was observed (Parks et al.,
1992). The presence of the presumed 6K t peptide in
infected plants has not yet been observed for any
potyvirus.
To gain insight into the nature of the A heptapeptide
* Author for correspondence. Fax +34 1 5854506. e-mail
[email protected]
1 Present address: California Institute of Technology, Division of
Biology 156-29, Pasadena, CA 91125, USA.
0001-2832 © 1995 SGM
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952
Short communication
1
10000
5000
I
I
I
I
P1
HC-Pro
I
I
I
TT
HC-Pro
I
I
P3
o,"
°o*
6K 1
o,
",,
CI
TT
v
6K 2
NIa
T
NIb
.:....
CP
,.. N"V V V H QTA"D'~ ,,,
In vitro
%,
-6
T
",
**-"
1~+1
Infectivity
Symptoms
tPpVW'
~i~i..- Q VVV H Q S K R ""i~
Yes
+++
tPPVQIII6H
~ii;'"
Yes
-
t P P V QIIlIN'slI17A
~iii...r~v
Yes
+
tPPvQmIN
~iii'"
I~llv V V H QTS K R . . . . . . ~
Yes
+
.... Q V V V H QTt~IK R " " i i i ~
Yes
++++
tPpVSt'lTA
I
aa
1
Q V V V Hr.lh K R . . - i i i ~
......
v v H
I
1111
I
Nla
Q
P1
I
QTI~IKR " " i i i ~
I ......
1119
processability
-----I
3140
Fig. 1. Schematic diagram of the mutations introduced into the sequence of the NIa cleavage site A of pGPPV and summary of the
results obtained after inoculation of N. clevelandii plants with the corresponding in vitro transcripts. The potyvirus genome is
represented in the upper part of the figure; the poly(A) tail is shown as a black rectangle and the VPg as a black circle; the protease
responsible for each proteolytic cleavage is indicated. The amino acid sequence at the A and F cleavage sites, as well as the different
mutations introduced into the site A sequence, are shown. The presence or absence of symptoms in the inoculated plants is indicated
(the number o f ' + ' signs correlating with the severity of the symptoms). The extent of the in vitro processability of the different NIa
site A mutant sequences is indicated; ' + ', partial cleavage; ' - ', no cleavage. This is a summary of the results presented in Garcia et
al. (1992).
as an in vivo NIa cleavage site and to determine whether
or not its processing is an essential step in the virus lifecycle, we have introduced several mutations into a fulllength PPV cDNA clone (from which infectious transcripts can be synthesized; Riechmann et al., 1990) that
alter the PPV A heptapeptide. These mutations had been
previously used to determine the partial processing in
vitro of PPV site A (Garc/a et al., 1992). The Q1116H
mutation changes the highly conserved glutamine residue
at position - 1 (see Fig. 1); it was subsequently shown
that this change resulted in a heptapeptide that was
unable to be processed in vitro. The Q1111N and S1117A
exchanges transform, when introduced together
( Q l l l l N , S l l l 7 A mutant), the sequence of the A
heptapeptide into that of the F heptapeptide (Fig. 1). The
natural NIa cleavage site F (NIb-CP junction) is very
efficiently cleaved, both in vitro and in vivo. However,
these changes only slightly affected cleavage at the NIa
site A.
These mutations (for details of the construction
procedure, see Garc/a et al., 1992) were introduced into
the full-length PPV clone pGPPV (Riechmann et al.,
1990) by fragment replacement, creating plasmids
p G P P V Qlll6H, p G P P V q n n ~ ' s l l x 7 A , p G P P V Q1111z~ and
pGPPV smTA (Fig. 1). RNA synthesis from linearized
plasmid templates and inoculation of young Nicotiana
clevelandii plants with the resulting capped transcripts
were performed as previously described (Riechmann et
al., 1990, 1991); the transcripts produced in vitro from
pGPPV and other cDNA clones were given the prefix 't'
(i.e. tPPV). N. clevelandii plants inoculated with tPPV
(wild-type) transcripts began to develop typical systemic
symptoms in the uninoculated upper leaves 7-10 days
after inoculation (Riechmann et at., 1990). All of the 10
plants inoculated with PPV Ql116n mutant transcripts
remained asymptomatic during a period of 31 days postinoculation (d.p.i.). To test whether the Q l l l 6 H
exchange was a lethal mutation that made tPPV Q111~H
unable to replicate (and therefore unable to establish an
infection), or was not lethal but hampered in some way
the normal virus life-cycle (since no symptoms were
detected), crude RNA samples from upper leaves of
plants inoculated with tPPV Qm6H were prepared and
subjected to RT-PCR as described previously
(Riechmann et at., 1991). The oligodeoxynucleotides
used as primers for the detection of viral RNA sequences
were: primer a, 5' AACAGCCTGGTCGAC 3' (complementary to positions 3627 to 3641 of PPV RNA;
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(a)
PPV ~,
RNA~
P3
t
6K l
i
I
~ -3'
a
50 nt
nt
600--
q
i-5 i-5 - ~
3i
15
15 15
d.p.i.
450 - -~--- 367 nt
270 - -
170 - 1
2
3
4
5
6
7
8
(b)
CP
/.,++ ,...+b..++..,-+o,
15
15
i
I
1
2
15
15
15
o
~
~
Ii
4
5
6
7
15
d.p.i.
"
~aJ
150 ng
31
3
8
0.83 mg leaf tissue
Fig. 2. (a) Detection o f viral R N A by R T - P C R in N. clevelandii plants
inoculated with PPV NIa site A mutant transcripts. PCR amplifications
were carried out with crude samples of R N A prepared from
uninoculated upper leaves (harvested 15 or 31 d.p.i., as indicated) of
plants inoculated with transcripts tPPV (wild-type, lane 3), tPPV qn16n
(lanes 4 and 5), tPPV qmlz~,sm~A (lane 6), tPPV Q1]IIN (lane 7),
tPPV smTA (lane 8) and with an extract of a mock-inoculated plant
(lane 2)• A control PCR using pGPPV as template is included (lane 1).
The region of PPV R N A that was amplified is represented; the
positions of the two oligodeoxynucleotides (a and b) used as primers
for PCR amplification are shown• (b) Detection o f PPV CP in N.
clevetandii plants inoculated with PPV NIa site A mutant transcripts.
Extracts were prepared from uninoculated upper leaves (harvested 15
or 31 d.p.i., as indicated) of plants inoculated with transcripts tPPV
(wild-type, lane 2), tPPV Qln~H (lanes 3 and 4), tPPV QnnN'slmA (lane
5), tPPV ° n n ~ (lane 6), tPPV smTA (lane 7) and from leaves of a mockinoculated plant (lane 8). A control sample of purified PPV CP (150 ng)
is included (lane 1). The amount o f leaf tissue corresponding to the
samples is indicated in the lower part o f the figure.
953
Fig. 2 a) and primer b, 5' CTCAATGATCAATGAGC 3'
(nt 3275-3291 ofPPV RNA; Fig. 2a). No viral RNA was
detected in any of the samples prepared from leaves
harvested 15 d.p.i. (Fig. 2a, lane 4 and data not shown).
However, viral sequences were amplified from two
extracts of leaves harvested at 31 d.p.i. (Fig. 2a, lane 5)
indicating that in these tPPVqll16H-inoculated plants the
virus RNA had systemically propagated. In accordance
with this result, PPV capsid protein (CP) could not be
detected in Western blots at 15 d.p.i. (Fig. 2b, lane 3),
but high levels of CP were present in these asymptomatic
plants 31 d.p.i. (Fig. 2 b, lane 4; immunological detection
of PPV CP was performed as previously described;
Riechmann et al., 1990). The R T - P C R products (that
correspond to the whole 6K1 peptide and the C terminus
of P3 protein; see Fig. 2a) derived from systemically
infected leaves, as well as a product obtained from the
inoculated leaves of one of these plants (also harvested at
31 d.p.i.), were sequenced using the primers that were
employed for their synthesis and the f m o l DNA
Sequencing System (Promega), essentially according to
the supplier's instructions. The Q l l l 6 H mutation was
present in the RNA sequence amplified from inoculated
leaves, as well as in those obtained from systemic leaves.
Surprisingly, an additional mutation that must have
been introduced in vivo during virus replication was
detected (Fig. 3a and data not shown). This second
mutation, a G to A exchange at nucleotide position 3513,
was detected in the inoculated leaf, although the wildtype G residue was predominant in the population of
PPV genomes, as inferred from the intensity of the
respective bands in the autoradiogram (Fig. 3 a). However, in the samples from systemically infected leaves, the
second-site mutation (A residue) was highly predominant
over the wild-type nucleotide, indicating that most of the
RNA molecules that were present in these leaves carried
this additional mutation (Fig. 3 a and data not shown)•
This nucleotide change at position 3513 results in the
substitution of a threonine for an alanine residue, seven
amino acids downstream from the scissile bond of the
NIa A site (Fig. 3 a). No other exchanges were detected
in the sequenced region.
N. clevelandii plants were inoculated with a crude
extract prepared from systemic leaves of two plants in
which the Q1116H,A1123T second mutant was detected.
Symptoms identical to those induced by wild-type PPV
developed in these plants (data not shown), suggesting
that the pathogenicity of the virus, which had been
greatly diminished by the Q l l l 6 H exchange, was
restored during its replication in vivo. The progeny virus
present in these plants, and those derived from one more
round of infection/replication, were analysed as above.
In most cases, both Q1116H and A1123T changes were
detected together in the same sample. None of the single
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954
Short communication
(a)
Plant 10 (31 d.p.i.)
Inoculated
leaf
AC
(b)
39.4 k D a 45.2 kDa
54.5 kDa
Systemic
leaf
GT
AC
GT
pGGP3AVwt
,; T
°°°°°
A
A
G/_.~A--nt3513
\
P3
"1
RV
6KIICI
A
.../
-"\
ff
A B
~
Q V V V H QTS...Q
pGGP3AVQ1116n
pGGP3AvQII16H,A1123T
A N
Q v v v al~ls...~lii
~
A
"\A
:C
--
+
-
+
-
+
! ii~z ~i~¸~
I ...CATC~AGC
Mutant
Ql116H
I!
~-A S
-1
nt 3513
I
AAG A G A GAC
g
R
D
TCA CAA GCA AAT...
S
Q
&
N
!
+1
MutantI---CAT~:GC AAGAGAGACTCACAA~CAAAT...
Qlll6H
progeny
[]
-1
S
K
+1
R
D
S
Q
a
i
~{i
N
1
2
I
4
5
6
Fig. 3. (a) Identification of the second-site mutation introduced into the 6K~ coding region of PPVQlneH progeny during replication
in N. clevelandii plants. Crude samples of RNA were prepared from both inoculated and uninoculated (systemically infected) leaves
of plant 10, removed 31 d.p.i. Viral sequences were then amplified by RT-PCR and sequenced. Autoradiograms showing the sequence
around the position (nt 3513) in which the newly introduced mutation (indicated by arrows) was found are presented, with the
nucleotide change indicated. The nucleotide and amino acid sequence of these regions in PPV Qmnn mutant and PPVQ~eH mutant
progeny are shown: residue changes derived from the site-directed mutagenesis procedure are shown within white squares, while the
mutation that was introduced in vivo (A1123T amino acid change) is highlighted in black. (b) Effect of the A1123T change on the in
vitro proteolytic processing of the NIa site A. A scheme of the plasmids employed in these experiments is shown: PPV ORF is
represented as an open bar; RV is the EcoRV site used to linearize the plasmid prior to transcription from the T7 promoter (black
circle). The expected molecular masses of the in vitro translation products and of the N-terminal fragments derived from proteolytic
processing at the different sites are indicated above the map. In vitro transcription/translation products from the indicated plasmids
were incubated with extracts of E. coli cells that did not ( - ) or did (+) express PPV NIa protease. The proteins were detected by
fluorography after SDS-PAGE on a 12.5% gel.
m u t a t i o n s was ever d e t e c t e d in the p r o g e n y analysis in
the a b s e n c e o f the other. These d a t a , t o g e t h e r with the
identification o f the A 1 1 2 3 T exchange in the two p l a n t s
p r i m a r i l y i n o c u l a t e d w i t h t P P V Q111~rt t h a t b e c a m e infected, suggest t h a t this s e c o n d m u t a t i o n c o u l d be
involved in the recovery o f the p a t h o g e n i c i t y o f the virus
( a l t h o u g h a d d i t i o n a l m u t a t i o n s in o t h e r regions o f the
P P V g e n o m e m i g h t have o c c u r r e d d u r i n g the infection
process). Occasionally, reversion to the w i l d - t y p e sequence at b o t h p o s i t i o n s was observed. These r e v e r t a n t s
m i g h t h a v e a l r e a d y arisen in the o r i g i n a l l y i n o c u l a t e d
p l a n t s a n d t h e n o u t c o m p e t e d the in v i v o - g e n e r a t e d
m u t a n t b y selection p r e s s u r e f a v o u r a b l e to w i l d - t y p e
RNA.
T o test if the A 1 1 2 3 T second-site m u t a t i o n p r o d u c e d
a recovery o f the in vitro p r o c e s s a b i l i t y o f the Q l l l 6 H
m u t a n t site A , b o t h c h a n g e s were i n t r o d u c e d t o g e t h e r in
p l a s m i d p G G P 3 A V wt ( G a r c i a et al., 1992), c r e a t i n g
p l a s m i d p G G P 3 A V QIxl~r~,Allz3T (Fig. 3b). T r a n s l a t i o n in
a r a b b i t reticulocyte lysate o f t r a n s c r i p t s synthesized
f r o m these p l a s m i d s resulted in a p o l y p e p t i d e with
e l e c t r o p h o r e t i c m o b i l i t y c o r r e s p o n d i n g to its expected
size (54"5 k D a ; Fig. 3b, lanes 1, 3 a n d 5). Processing a t
sites A o r B w o u l d give rise to N - t e r m i n a l f r a g m e n t s o f
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Short communication
39-4 and 45"2 kDa, respectively, and polypeptides with
these electrophoretic mobilities were readily detected
when the translation products derived from tGGP3AV wt
were incubated with an extract of E. coli cells that
express the NIa protease (Fig. 3b, lane 2; proteolytic
processing assays were performed as previously described; Garcia et al., 1992). The histidine for glutamine
substitution at position - 1 of the A heptapeptide present
in products derived from tGGP3AV qm~rI precluded the
generation of the 39.4 kDa band after the proteolytic
incubations (Fig. 3b, lane 4; Garcia et al., 1992). The
processability of this mutant heptapeptide was not
rescued by the second mutation Al123T, since the
39.4 kDa band was not generated from products of
tGGP3AV Q1116n'Al123T either (Fig. 3 b, lane 6).
N. clevelandii plants were also inoculated with
t P P V Q1111•'s1117A, tPPV Q1111z~ and tPPV sll17A. While
tPPVQllllN'S1117Aand tPPV Q1111z~ had a reduced pathogenicity in view of the extremely mild symptoms that
they induced, t P P V s1117A mutant was more virulent than
tPPV wt (data not shown). The time course of symptom
development was also altered by this mutation;
tPpVSmTA-inoculated plants developed symptoms 5-7
d.p.i. (i.e., earlier than plants inoculated with wild-type
transcripts). Viral RNA was readily detected by RTPCR at 15 d.p.i, in systemic leaves from plants inoculated
with these mutants (Fig. 2a, lanes 6, 7 and 8). Similar
high levels of CP were detected in plants inoculated with
each of these three mutants and with tPPV wt, indicating
that the differences in the severity of the induced
symptoms apparently did not correlate with differences
in virus accumulation (Fig. 2b, compare lanes 2, 5, 6 and
7).
As above, viral RNA sequences were amplified from
systemic leaves and the PCR products were sequenced.
The presence of Q l l l l N , S l l l 7 A , Q l l l l N and S l l l 7 A
mutations in the corresponding viral progeny was
confirmed. N o in vivo-introduced mutations were
detected in the sequenced region. Progeny virus of these
three mutants maintained the corresponding parental
phenotype with regard to the symptom induction
process.
All these data indicated that the
Q l l l l N , S l l l 7 A , Q l l l l N and SII17A mutations were
stable when in the PPV genome.
As described above, the development of a normal PPV
life-cycle was hampered by the Q l l l 6 H mutation, yet
PPV Ql116Hmutant was able to establish an infection in N.
clevelandii plants, indicating that a cleavable NIa site A
is not an essential requirement for virus viability. This
conclusion relies on the assumption that the Q l l l 6 H
mutation abolishes in vivo processing of the PPV site A
in a similar way as it does in vitro (Garcia et al., 1992),
but such assumption is warranted by the current
abundance of data on potyvirus proteolytic processing.
955
Changes at the highly conserved - 1 position have been
engineered in several cleavage sites of different potyviruses and their effects assayed in various systems. They
have always been found to abolish the cleavability of the
resulting mutant sites (Dougherty et al., 1988, 1989;
Garcia et al., 1989a). In addition, a replacement of the
glutamine residue present at the - 1 position of the NIa
site E (NIa-NIb junction) (a change similar to Q1116H
that also abolishes in vitro cleavability; Garcia et al.,
1989a) was introduced into the PPV genome and the
resulting mutant transcripts were unable to establish an
infection in N. clevelandii plants (data not shown).
Mutation of the - 1 glutamine residue of the TEV NIa
site D (6K~-NIa junction) also eliminates virus viability
(Restrepo-Hartwig & Carrington, 1994). These results
indicate the capability of - 1 glutamine mutations to
render viral genomes non-viable when introduced into in
vivo-functional NIa sites, and therefore reinforces the
interpretation of the processing at NIa site A as somehow
dispensable. This dispensability would imply that the
full-length product (i.e. P3+6KI) is the functional
protein. On the other hand, the fact that the
Q l l 11N,S1117A, Q l l l l N and S l l l 7 A (as well as other
mutations not described in this article; J. L. Riechmann
& J.A. Garcia, unpublished results) were viable and
stable suggests that a certain degree of tolerance towards
amino acid exchanges exists in the heptapeptide and
surrounding sequence when a cleavable site is conserved.
In vivo processing of the A site in the normal life-cycle
of PPV could be a way to regulate the activity (currently
unknown) of the P3 + 6K1 protein. Such a regulatory
role for NIa site A processing seems to be appropriate if
the characteristics of this part of the polyprotein are
considered, since this site precisely defines the boundary
between a region (P3) with a very low level of sequence
similarity among different potyviruses and another one
(6K~) that is much more conserved (Lain et aL, 1989; for
additional potyvirus genomic sequences see Johansen et
al., 1991; Bowman Vance et al., 1992; Jayaram et al.,
1992; Nicolas & Lalibert6, 1992; Yeh et al., 1992; Gough
& Shukla, 1993; Puurand et al., 1994; Gunasinghe et al.,
1994). Being the function of the NIa site A to provide a
regulatory step, it is understandable that this site be
present in all the potyviruses sequenced to date. The fact
that cleavage at the TEV site A was not detected in vitro
(Parks et al., 1992) could also fit into this model, since
TEV may have evolved to make this level of control
dispensable. Interestingly, the TEV NIa site A is less well
conserved (when compared to the rest of TEV NIa
cleavage sites) than those A sites of the rest of potyviruses
for which data are available (Riechmann et al., 1992;
Garcia et al., 1992).
In summary, the fact that tPPV °l~a6n was able to
initiate an infection indicates that a cleavable NIa site A
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956
Short communication
is not an essential requirement for such a process.
However, the drastic effects that mutations at this
cleavage site have on the course of virus infection, as well
as the appearance of a second mutation and the reversion
to the wild-type sequence, suggest that processing at the
P 3 - 6 K 1 junction plays a relevant role in the life-cycle of
PPV and that proper cleavage contributes to the timecourse of symptom development and severity.
We are grateful to Dr F. Garcia-Arenal (E.T.I.A., Madrid) for
greenhouse facilities and to Drs E. Rodriguez-Cerezo and B. Krizek for
valuable comments on the manuscript. This work was supported by
Grants BIO92-0187 from CICYT and C168/90 from Comunidad
Aut6noma de Madrid, and by an institutional grant from Fundaci6n
Ram6n Areces to the Centro de Biologia Molecular. J.L.R. was the
recipient of a postdoctoral fellowship from C.S.I.C. and M.T.C. was
recipient of a fellowship from M.E.C.
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(Received 11 August 1994; Accepted 13 December 1994)
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