From the Laboratoire de rétrovirologie humaine, Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montréal, Québec H3C 3J7, Canada
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ABSTRACT |
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The 96-amino acid Vpr protein is the major
virion-associated accessory protein of the human immunodeficiency virus
type 1 (HIV-1). As Vpr is not part of the p55 Gag polyprotein precursor (Pr55gag), its incorporation requires an anchor to associate
with the assembling viral particles. Although the molecular mechanism
is presently unclear, the C-terminal region of the Pr55gag
corresponding to the p6 domain appears to constitute such an anchor
essential for the incorporation of the Vpr protein. In order to clarify
the mechanism by which the Vpr accessory protein is
trans-incorporated into progeny virion particles, we tested whether HIV-1 Vpr interacted with the Pr55gag using the yeast
two-hybrid system and the maltose-binding protein pull-down assay. The
present study provides genetic and biochemical evidence indicating that
the Pr55gag can physically interact with the Vpr protein.
Furthermore, point mutations affecting the integrity of the conserved
L-X-S-L-F-G motif of p6gag completely abolish the
interaction between Vpr and the Pr55gag and, as a consequence,
prevent Vpr virion incorporation. In contrast to other studies,
mutations affecting the integrity of the NCp7 zinc fingers impaired
neither Vpr virion incorporation nor the binding between Vpr and the
Pr55gag. Conversely, amino acid substitutions in Vpr
demonstrate that an intact N-terminal Unlike simple retroviruses,
HIV-11 encodes for regulatory
(tat and rev) as well as for accessory
(vpr, vpu, vif, and nef)
genes, together referred to as the auxiliary genes. The tat
and rev regulatory genes have been shown to be absolutely
essential for viral replication in vitro (1). Recently, an
increasing number of studies demonstrate that mutations affecting the
other auxiliary genes, called the accessory proteins, cause significant
phenotypic defects in HIV-1 replication, suggesting that these
accessory genes may play pivotal roles during in vivo
infection and pathogenesis (2).
The Vpr accessory gene product encodes a 14-kDa, 96-amino acid nuclear
protein that is expressed late during viral replication in a
Rev-dependent manner (3). This protein is highly conserved between HIV-1, HIV-2, and simian immunodeficiency virus (SIV). In
addition, HIV-2 and SIVs encode for a protein, Vpx, that has been shown
to possess many structural as well as functional similarities with the
Vpr protein (4). Functionally, the HIV-1 Vpr protein harbors two main
biological activities. First, early during infection of nondividing
cells, Vpr is implicated in the nuclear translocation of the
preintegration complex (5, 6). The mechanism by which Vpr influences
the transport of the preintegration complex remains unclear. Although
no classical nuclear localization signal have been clearly demonstrated
in Vpr, it is likely that Vpr acts through interactions with cellular
proteins involved in the nuclear import of macromolecules. In fact, it
was recently demonstrated that Vpr could associate with importin- An important feature of the HIV-1 Vpr and the HIV-2/SIV Vpx proteins is
that they are selectively incorporated into the virus particles, which
indeed suggests an early function for these two proteins during the
viral life cycle (19, 20). The localization of Vpr and Vpx within
virions is still unclear. Immunoelectron microscopic studies suggested
that the HIV-1 Vpr protein localized beneath the viral envelope,
co-localizing with the Gag p24 core structures (21). However, a more
recent analysis by Kewalramani and Emerman (22) placed the Vpx protein
within HIV-2 cores. As Vpr and Vpx are not synthesized as part of the
Gag or Gag-Pol polyprotein precursors, they must utilize a distinct
mechanism in order to be incorporated into virion particles.
Lavallée et al. (23) reported that Vpr could be
specifically incorporated in trans within virus-like
particles originating only from the expression of the
Pr55gag. The C-terminal p6 domain of the
Pr55gag was subsequently demonstrated to be
essential for the incorporation of Vpr into virus particles (24, 25).
Furthermore, the integrity of a very conserved motif in the C-terminal
region of the p6 domain, L-X-S-L-F-G, was shown to be
critical for Vpr virion incorporation (26, 27). Previous studies
clearly established that the predicted amphipathic In order to clarify the mechanism by which the HIV-1 Vpr protein is
trans-incorporated into virion particles, we used
protein-to-protein interaction assays to investigate Vpr-Gag
interactions. The present work provides genetic (yeast two-hybrid) and
biochemical evidence indicating that the incorporation of Vpr into
HIV-1 particles involves a direct association between Vpr and the p55
Gag polyprotein precursor. We demonstrate that the p6 domain is
necessary and sufficient for this interaction. The direct binding
of Vpr to the p55 precursor may constitute a target for the development of molecules that could prevent Vpr virion incorporation, and thus, Vpr
early functions.
Bacterial and Yeast Strains--
Manipulations of bacterial
strains and of DNAs were performed by standard methods (34) unless
otherwise noted. Escherichia coli AG1-competent cells were
used for routine DNA manipulations. Yeast strain EGY48 (MATa,
trp1, ura3, his3, leu2::plexAop6-leu2) was
used as a host strain for all two-hybrid experiments and was obtained
from the laboratory of Dr. Roger Brent (Massachusetts General Hospital,
Boston, MA).
Construction of Plasmids--
To construct the bait plasmids
LexA-Vpr, LexA-Vpu, and LexA-Pr55gag, the
vpr and gag genes from the HxBRU provirus plasmid
(35) and the vpu gene from the HxBH10 (36) provirus plasmid
were amplified by polymerase chain reaction (PCR) using the 5' primer 5'-GGCCTAAGGACTGGGTACGATCAA-3' and the 3' primer
5'-GACTTTCAGATAACGAATACTA-3' for Vpr, the 5' primer
5'-GAAGGAGAGGCATCCGTGCGAGAG-3' and 3' primer 5'-GAAGGAGAGGCATCCGTGCGAGAG-3' for Gag, the 5' primer
5'-GTAGTACATGGGATCCAACCTATACA-3' and 3' primer
5'-TCCTTCGGATCCAGTACCCCATAA-3' for Vpu, all containing BamHI sites. The PCR fragments were then cloned in this
latter restriction site in translational frame with the codons of the LexA DNA binding domain of the pEG202 vector (37). These latter bait
fusion proteins were produced constitutively from pEG202, a 2-µm
HIS3+ plasmid under the control of the
ADH1 promoter and encoding the LexA C-terminal
oligomerization region, which contributes to the operator occupancy by
LexA derivatives (37). To construct LexA-Vpr mutants, such as
LexA-VprE25K, LexA-VprA30F, LexA-VprQ65E, LexA-VprSR79-80ID, and
LexA-VprR80A, the similar Vpr PCR fragments amplified from HxBRU
harboring the different Vpr mutants, E25K, A30F, Q65E, SR79-80ID, and
R80A, were fused in frame with the LexA DNA binding domain of the
pEG202 vector. The design and construction of these mutants have been
described elsewhere (18, 29). The Gag BamHI fragment was
also cloned in the pET-21c expression vector (Novagen), which was used
to produce in vitro labeled
Pr55gag.
The prey plasmids B42-Vpr, B42-Pr55gag, and
B42-Vpu were constructed by digesting the Vpr,
Pr55gag, and Vpu cDNAs from pEG202 with
EcoRI and XhoI. These
EcoRI-XhoI fragments were placed in pJG4-5, a
2-µm TRP1 plasmid (38), in translational frame with the
codons for the simian virus 40 large T nuclear localization signal, the
B42 transactivation domain, and the hemagglutinin epitope tag. Because
the pJG4-5 vector is under the control of the GAL1
promoter, the expression of the prey fusion proteins was inducible in
yeast grown on minimal medium containing 2% galactose and 1%
raffinose (Gal/Raff) but not in yeast grown on 2% glucose (Glc).
Construction of the Moloney murine leukemia virus (MLV) Gag/HIV-1 p6
chimeric construct and the HIV-1 Pr55gag (p6
L44P/F45S) p6 double mutant were described previously (25, 39). As
well, the design and construction of the NCp7 mutants were described
elsewhere: H23C (31), C28S/C49S (23), and
To generate Vpr and Vpu expression plasmids, the vpr and
vpu genes from the HIV-1 ELI isolate were amplified by PCR
using the 5' primer 5'-AGAGTCGACGAACAAGCCCCAGCAGAC-3' and the 3' primer 5'-GGCCTGCAGTTAGGATCTACTGGATCC-3' for Vpr and the 5' primer
5'-CATGTCGACCAACCTTTAGGGATAATA-3' and the 3' primer
5'-TCTCTGCAGCTACAGGTCATCAATATC-3' for Vpu. The PCR products were then
directly cloned into the EcoRV restriction site of the
pBluescript KS+ vector (Stratagene). Digestion of pBKS+/Vpr and
pBKS+/Vpu with SalI yielded Vpr and Vpu fragments lacking
their initial methionine codon and were placed into the SalI
restriction site of the pMAL-c2 vector (New England Biolabs). This
placed amino acids 2-96 of Vpr and amino acids 2-81 of Vpu into
translational phase with maltose-binding protein (MBP) sequences of the
pMAL-c2 vector.
Transformation of Strain with Reporter, Bait, and Prey
Plasmids--
The selection strain were made by transforming the EGY48
yeast strain with a URA3 lacZ ( Determination of Bait-Prey Interaction--
Five independent
transformants containing the appropriate bait and prey plasmids were
streaked on Glc Ura In Vitro Binding Studies--
To produce MBP, MBP-Vpu, and
MBP-Vpr proteins, E. coli XL1-BLUE cells (New England
Biolabs) transformed with pMAL-c2, pMAL-c2/Vpu, or pMAL-c2/Vpr plasmids
were cultured in M9 medium (0.2 M NaCl, 10 mM
MgSO4, 0.1 mM CaCl2, 0.5% casamino
acids, 0.5% glucose, 0.2 mM thiamine, and 0.1 mg/ml
ampicillin). Protein expression was then induced by adding
isopropyl-1-
To investigate Vpr-Gag interaction, we first prepared MBP, MBP-Vpu, and
MBP-Vpr-bound amylose resin by incubating equivalent levels of MBP,
MBP-Vpu, and MBP-Vpr proteins with amylose resin (50% v/v) for 60 min
at 4 °C. Then, equal amounts of in vitro synthesized
[35S]methionine-labeled Pr55gag
(TNT coupled reticulocyte lysates system; Promega) diluted in Column
Buffer (20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA) were incubated with MBP, MBP-Vpu, or MBP-Vpr-bound
amylose resin for 2 h at 4 °C. These complexes were then washed
several times with Column Buffer, and bound proteins were eluted with
10 mM maltose, loaded onto a 12.5% SDS-PAGE for
autoradiography or Western blot analysis.
Immunoprecipitation and Western Blot--
Yeast EGY48 cells
expressing B42-Pr55gag and LexA-Vpr, which was
used in yeast two-hybrid assay, were lysed in 500 µl of RIPA lysis
buffer (41) by beating with glass beads five times for 2 min each.
After being removed from beads and cell debris by centrifugation
(10,000 × g) at 4 °C, the specific proteins in the
cell lysates were immunoprecipitated with either a rabbit anti-Vpr
serum (23) or a mouse anti-p24 antibody (ATCC HB-9725). Immunoprecipitated proteins were then subjected to SDS-PAGE and subsequently blotted on nitrocellulose (0.45 mm; Schleicher & Schuell).
LexA-Vpr and B42-Pr55gag fusion proteins were
identified by the rabbit anti-Vpr serum, or a rabbit anti-p24 antibody
(NIH 384), respectively, with highly sensitive ECL chemiluminescence
detection system as recommended by the manufacturer (Amersham Pharmacia Biotech).
Radioimmunoprecipitation and Virion Incorporation
Assay--
Virion incorporation assay was performed as described
previously with slight modifications (29). Briefly, for the HIV-1 Pr55gag (p6 L44P/F45S) mutant, MT-4 cells
(5 × 106) were transfected using the DEAE-dextran
method with either 10 µg of wild-type provirus constructs (pNL4.3) or
provirus harboring the Pr55gag (p6 L44P/F45S)
mutation. 48 h after transfection, cells were metabolically
labeled with 200 µCi of [35S]methionine for 12 h.
Radiolabeled cells and 20% sucrose cushion-pelleted virions were lysed
in RIPA buffer (10 mM Tris-HCl (pH 7.4), 1 mM
EDTA, 100 mM NaCl, 1% (v/v) Triton, 0.2% (w/v)
phenylmethylsulfonyl fluoride) and immunoprecipitated with a mix of
rabbit anti-Vpr and HIV-1-seropositive human serum, as described
previously (29). The immunoprecipitated complexes were loaded on a
12.5% (w/v) SDS gel and analyzed by autoradiography. To test virion
incorporation of Vpr for the HIV-1 p7 H23C and HIV-1 p7 Genetic Evidence That Vpr and the Pr55gag Physically
Interact--
The experimental system described by Golemis et
al. (37) was used to investigate the interaction between Vpr and
the Pr55gag of HIV-1. In order to do so, the Vpr
and p55 proteins were fused at the C terminus of the LexA DNA binding
domain (bait) and the B42 bacterial transactivator (prey). To monitor
the specificity of interactions, the native plasmids containing only
the LexA DNA binding domain or the B42 bacterial transactivator domain, or fused with another HIV-1 accessory protein, Vpu, which is unlikely to interact with HIV-1 Gag protein (42), were used as controls of
specificity. The host strain (EGY48) contains the LEU2 and the lacZ reporter genes, both carrying LexA operators
instead of native upstream regulatory sequences. A EGY48 yeast cell
containing a bait (LexA-fusion) plasmid and reporters (LEU2
and lacZ) remains inert for the expression of leucine
utilization or
Essentially, the yeast strain EGY48 was transformed with different
combinations of bait and prey plasmid constructions and selected on Glc
Ura
Finally, Western blot analysis was performed to confirm that the
LexA-Vpr bait and the B42-Pr55gag prey expressed
the expected fusion proteins (Fig. 1B). A 36-kDa LexA-Vpr
fusion protein was detected using a rabbit polyclonal anti-Vpr serum,
while the B42-Pr55gag fusion corresponded to a
68-kDa protein using a rabbit anti-p24 serum. Since the
B42-Pr55gag is a large fusion protein, the other
bands detected by Western blot analysis are likely to be degradation
products generated during yeast protein extraction.
Regions within the Pr55gag and Vpr Important for the
Interaction--
Previous reports clearly established that the p6
domain of the Pr55gag was necessary and
sufficient for Vpr virion incorporation (24, 25, 27). In order to
determine the domains of importance for the
Vpr-Pr55gag direct interaction, we first tested
different Gag constructs in the two-hybrid system (Fig. 2A).
Kondo et al. (25) showed that the addition of the p6 domain
to the C terminus of the MLV p65 Gag precursor was sufficient to
mediate Vpr incorporation, while the wild-type MLV Gag precursor was
incapable of incorporating Vpr (Fig. 2A). In order to
determine if this specific incorporation of Vpr into MLV/HIV p6
chimeric virus was based on a direct interaction between the chimeric
Gag gene product and Vpr, we quantitatively measured
de Rocquigny et al. (31) recently reported that the
integrity of the zinc finger structures in the NCp7 was important for Vpr virion incorporation. Consequently, we investigated the effect of
NCp7 mutants on the Vpr-Pr55gag interaction.
Fig. 2A demonstrates the structure of the NCp7 zinc fingers.
Different mutants affecting the zinc binding domains (H23C is a
substitution of His23 for Cys; C28S/C49S contains
substitutions of Cys28 and Cys49 for Ser;
We next wanted to investigate the region of Vpr that is important for
its association with the Pr55gag. In order to
address this question, five Vpr point mutants affecting different
structural regions of the protein were fused to LexA sequences (Fig.
3A) and used for interaction
experiments using the yeast two-hybrid system. The E25K and A30F
mutants affect the predicted N-terminal Direct in Vitro Interaction between Vpr and the p55 Gag
Precursor--
In order to confirm whether the association between Vpr
and the Pr55gag observed in yeast could be
reproduced using another approach, we took advantage of an in
vitro binding assay using recombinant fusion proteins. First, the
vpr gene was introduced into the pMAL-c2 vector in fusion
with the maltose-binding protein (MBP). MBP, MBP-Vpu, and MPB-Vpr
fusion proteins were then produced in bacteria and purified as
described under "Experimental Procedures." Then, the entire Gag
open reading frame was used to generate in vitro labeled
protein. The in vitro translated
Pr55gag was incubated with MBP, MBP-Vpu, or
MBP-Vpr fusion proteins, that were previously immobilized on amylose
resin. Following a 2-h incubation, the complexes were washed several
times, eluted, and then analyzed on a 12.5% SDS-PAGE. As shown in Fig.
4A, the Pr55gag was able to specifically interact with
MBP-Vpr, and not with MPB-Vpu nor with MBP alone. To ensure that the
55-kDa band associating with MBP-Vpr was really the p55 Gag precursor,
we electrotransferred the SDS gel onto a nitrocellulose filter and
performed a Western blot using an anti-p24 antibody. The immunoblot
confirmed that the 55-kDa protein was effectively the
Pr55gag (data not shown). An additional 32-kDa
band also specifically associated with the MBP-Vpr fusion protein (Fig.
4A, lane 1). This fragment was
confirmed to be a Gag-related product by Western blot analysis (data
not shown). The smaller proteins in the input loading (lane
4) probably represent nonspecific cleavage products, products from initiation at downstream AUG, or premature translation termination.
We next attempted to analyze the strength of the Vpr-Gag interaction by
studying the effect of increasing salt concentration on the recovery of
the Pr55gag by the MBP-Vpr proteins. This
technique was previously used to determine the strength of the
interaction between the cyclophilins A and B and the Gag protein (48).
As demonstrated in Fig. 4B, the
Vpr-Pr55gag interaction could sustain 900 mM NaCl. Coomassie Blue staining of the gel demonstrated
that equal amounts of MBP-Vpr were still present after washing with
these different salt concentrations (data not shown). Together, these
results demonstrate biochemical evidence for a direct binding between
the Vpr protein and the Pr55gag, and confirm the
data demonstrated genetically using the yeast two-hybrid system (Fig.
1). Furthermore, we conclude that the association between the
Pr55gag and Vpr is a strong interaction in
vitro, sustaining salt concentrations of 900 mM NaCl.
We next wanted to determine if we could interfere with the interaction
by specifically blocking the accessibility of the
Pr55gag to the MBP-Vpr fusion protein. In order
to address this question, we used two different anti-Vpr antibodies: a
polyclonal rabbit antiserum generated against recombinant Vpr protein
(23) and a rabbit polyclonal anti-peptide serum that recognizes the
first 19 amino acids of Vpr (46). As controls, anti-Vpu and anti-Myc antibodies were used for the experiments. These different antibodies were incubated for 3 h in the presence of immobilized MBP-Vpr, washed several times to remove unbound and nonspecifically attached antibodies, and then presented to equal amounts of
[35S]methionine-labeled Pr55gag.
Although the anti-Vpu and anti-Myc antibodies demonstrated slight nonspecific competition with the MBP-Vpr-Pr55gag
interaction, this nonspecific effect never reached the levels observed
with the anti-Vpr antibodies in several experiments. Interestingly, the
anti-Vpr antibody directed against the first 19 amino acids of Vpr
(lane 3) interfered more efficiently with the
MBP-Vpr-Pr55gag interaction than the polyclonal
anti-Vpr (lane 2). The result presented in Fig.
4C suggests that the binding between Vpr and p55 can be
affected and that the potential use of other molecules could be used to
affect the Vpr-Pr55gag interaction and
consequently prevent Vpr virion incorporation.
Mutations That Affect the Binding between Vpr and the
Pr55gag Also Affect Vpr Virion Incorporation--
In order
to confirm our hypothesis that direct binding between Vpr and the
Pr55gag is required for Vpr virion
incorporation, we investigated whether our mutants in the
Pr55gag were still capable of incorporating the
Vpr accessory protein into virions. To analyze the HIV-1
Pr55gag (p6 L44P/F45S) mutant for Vpr virion
incorporation, MT-4 cells were transfected with an infectious proviral
clone of HIV-1 (pNL4.3) expressing in cis either the
wild-type or the Pr55gag (p6 L44P/F45S) protein.
Following transfection, the cells were radiolabeled and the ability of
Vpr to be incorporated into virion was monitored by immunoprecipitating
both cell and virion-associated viral proteins. We have previously
described a ratio system to assess the amount of Vpr found in the
virions as a proportion of the total amount found in the cell using
other virion proteins as standards (RT) (29). Essentially, the
virion-associated Vpr value is calculated based on the level of
internal control p66 (RT) present in the virions as well as the
proportion of the total Vpr found in the cell as determined by
densitometric scanning of autoradiograms. This value is then compared
with wild-type virion-associated Vpr value, which is set at 100%. As
can be observed in Fig. 5A,
substantial amounts of Vpr could be detected when proteins from
wild-type virions were immunoprecipitated (lane 8). However, no detectable amount of Vpr protein was
observed in virions generated from the proviral construct harboring the p6 L44P/F45S mutation (Fig. 5A, lane
7), even though Vpr was expressed in transfected MT-4 cells
(Fig. 5A, lane 3). This observation is
consistent with our result (Fig. 2B) that this mutant in the context of the Pr55gag is incapable of binding
Vpr, thus suggesting that a direct interaction between Vpr and the p6
domain of the Pr55gag is required for Vpr virion
incorporation.
We previously demonstrated that proviral constructs harboring the HIV-1
p7 C28S/C49S mutations in the nucleocapsid region of Gag did not
extensively affect Vpr virion incorporation (23). This is consistent
with our binding analysis (Fig. 2B), suggesting that the
integrity of the zinc binding motifs of the NCp7 is not critical for
the interaction between Vpr and the Pr55gag. In
order to further confirm this, we decided to analyze the ability of Vpr
to incorporate in viruses harboring either the HIV-1 p7 H23C or the
HIV-1 p7 HIV-1 Vpr is the major virion-associated accessory protein. As Vpr
is not synthesized as part of the Gag polyprotein precursor, it must
utilize a distinct mechanism in order to be incorporated into virion
particles. It has been clearly demonstrated through Gag deletion
analysis that the virion incorporation of the Vpr protein requires the
p6 domain from the p55 precursor (24, 25, 27). However, the association
between Vpr virion incorporation and Vpr-Pr55gag
binding was still missing. Several groups (26, 27) demonstrated that
single amino acid substitutions or deletions of either
Leu44 or Phe45 in the p6 domain abolished the
ability of Vpr to be incorporated in the context of MLV/HIV p6 or Rous
sarcoma virus/HIV p6 chimeric viruses. In addition, the inability of
our HIV-1 Pr55gag (p6 L44P/F45S) double mutant
to incorporate Vpr was confirmed in the context of an infectious
proviral clone of HIV (Fig. 5A). Consequently, the inability
of Vpr to interact with the HIV-1 Pr55gag (p6
L44P/F45S) double mutant (Fig. 2B) brings a direct
correlation between the lack of direct binding to the
Pr55gag and the incapacity of Vpr to be
incorporated into virion particles.
Evidence of direct interaction between Vpr and other Gag domains has
been recently reported. de Rocquigny et al. (31) reported that the zinc fingers of the nucleocapsid protein (NC) were important for Vpr virion association. Moreover, using chemically synthesized peptides, they demonstrated that Vpr could directly interact with NCp7,
but not with p6 in vitro. However, our result demonstrated that only mutants in the p6 domain (LF-PS) resulted in the loss of
binding between Vpr and the Pr55gag, while
mutations or the complete deletion of the p7 zinc fingers did not
affect the interaction (Fig. 2B) and Vpr incorporation (Fig.
5B). It is possible that the zinc fingers of the NCp7 are important for Vpr binding in the context of the mature NC. However, our
results indicate that in the context of the
Pr55gag, the zinc finger motifs are less
critical for the Vpr-Pr55gag interaction.
Moreover, in contrast to de Rocquigny et al. (31), who
demonstrated significant defects in Vpr virion incorporation using NC
mutants, in particular with the HIV-1 p7 H23C mutant, our result did
not reveal extensive impairment of Vpr virion-incorporating ability
(Fig. 5, B and C). It is possible that this
discrepancy results from experimental differences. Indeed, the basis
for quantification of Vpr encapsidation are different, de Rocquigny's
group used Western blot while we used radioimmunoprecipitation.
Consequently, the importance of NCp7 in HIV-1 Vpr incorporation still
needs to be demonstrated. In fact, Wu et al. (49) deleted
the complete NCp8 sequence in HIV-2 and did not affect the ability of
Vpx to be incorporated into virions, while removal of p6 sequences
resulted in loss of Vpx incorporation.
The structural domains within Vpr important for incorporation have also
been studied. The extensive mutagenic analysis of several groups agrees
that the amphipathic From our and other groups' results, we present a model for the
molecular mechanism by which the Vpr protein is
trans-incorporated into progeny virions. We suggest that
HIV-1 Vpr can only associate with p6 in the context of the
Pr55gag. In this context, p6 would have a proper
conformation to directly associate with Vpr, and subsequently pull it
within forming virions. Then, upon activation of the viral protease,
the Pr55gag would be processed and mature p6
would be release from the Pr55gag. This release
of p6 would result in its dissociation from Vpr. Subsequently, Vpr
could associate with other virion proteins such as NC in the viral core
in order to fulfill its functional role in the context of the
preintegration complex early in infection.
In summary, the results presented here demonstrate direct interaction
between Vpr and Gag in the context of the p55 precursor. Furthermore,
our results suggest that Vpr trans-incorporation requires a
direct binding to the p6 domain of the Pr55gag.
The development of an assay that demonstrates this critical interaction may allow the screening of molecules that prevent Vpr virion
association and thus, Vpr early function, which could ultimately impair
HIV-1 infection.
-helical structure is
essential for the Vpr-Pr55gag interaction. Vpr and the
Pr55gag demonstrate a strong interaction in vitro
as salt concentrations as high as 900 mM could not disrupt
the interaction. Finally, the interaction is efficiently competed using
anti-Vpr sera. Together, these results strongly suggest that Vpr
trans-incorporation into HIV-1 particles requires a direct
interaction between its N-terminal region and the C-terminal region of
p6gag. The development of Pr55gag-Vpr interaction
assays may allow the screening of molecules that can prevent the
incorporation of the Vpr accessory protein into HIV-1 virions, and thus
inhibit its early functions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and the nucleoporin Nsp1, and thus possibly play the role of an
importin-
-like protein (7-9). Consistent with its involvement in
the nuclear targeting of the preintegration complex, Vpr was shown to
be required for efficient replication in nondividing cells such as
monocytes and macrophages (6, 10, 11). The ability of Vpr to arrest the cell cycle constitutes the second biological activity associated with
this protein (12, 13). The cytostatic effect of Vpr was shown to result
in a specific block in the G2 phase of the cell cycle,
which was correlated with the inactivation of the Cdc2 kinase (14, 15).
The functional role of Vpr-mediated cell cycle arrest in proliferating
and nondividing HIV target cells is still unclear. However, Stewart
et al. (16) recently demonstrated that Vpr could also induce
apoptosis following cell cycle arrest, suggesting a contribution to CD4
cell depletion during HIV-1 disease. As well, the cell cycle arrest
action of Vpr was shown to increase viral expression in dividing T
cells as well as in macrophages (11, 17, 18).
-helical
structure located within the N-terminal region of Vpr was important for
its packaging into virions (28-30). Based on the data accumulated so
far, the mechanism of Vpr incorporation into virion particles suggests
a direct interaction between the p6 domain of the p55 Gag precursor and
Vpr. Nonetheless, Vpr was also shown to associate with other domains
from the Pr55gag. The zinc fingers of NCp7 have
recently been suggested to be important for the virion incorporation of
Vpr (31, 32). In addition, evidence suggesting an association between
Vpr and the MAp17 has also been obtained (33).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
K14-T50 (40). The MLVGag,
MLVGag/HIV-1 p6, HIV-1 Pr55gag (p6 L44P/F45S) p6
double mutant, HIV-1 Pr55gag (p7 H23C), HIV-1
Pr55gag (p7 C28S/C49S), and HIV-1
Pr55gag (p7
K14-T50) were amplified by PCR
using the following primer sets: MLV gag: 5' primer
5'-GCCGCGGATCCGCCAGACTGTTACCACTCCC-3'; 3' primer
5'-GCAAGGATCCTAGTCATCTAGGGTCAGGAG-3'. MLVGag/HIV-1-p6: 5' primer
5'-GCCGCGGATCCGCCAGACTGTTACCACTCCC-3'; 3' primer
5'-GCGCGCCTAGGTCTTTATTGAGTAGCGGG-3'. HIV-1
Pr55gag (p6 L44P/F45S) mutant, and the HIV-1
Pr55gag (p7 H23C), (p7 C28S/C49S), (p7
K14-T50) mutants: 5' primer GCCGCGGATCCGTGCGAGAGCGTCAGTATTA-3'; 3'
primer CGGCGGGATTCTCTTTATTGTGACGAGGG-3'. The PCR fragments were
digested with BamHI and cloned in the pEG202 vector. These fragments were then taken out of pEG202 by digesting with
EcoRI and SalI, and subcloned in the
EcoRI-XhoI restriction sites of the pJG4-5 vector.
-galactosidase) reporter
plasmid and the different HIS3 bait plasmids by the lithium
acetate method (34). The yeast selection strain harboring the bait and
reporter plasmids were transformed with different prey plasmid DNAs,
and tryptophan utilization phenotype was used (in addition to His and
Ura markers for bait and LacZ reporter plasmids,
respectively) for selection of transformants with the prey plasmids.
His
Trp
medium for amplification. Two days later, transformants were restreaked
on plates containing Glc Ura
His
Trp
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-Gal)
medium or Gal/Raff Ura
His
Trp
X-Gal medium to assess transcriptional activation of
the lacZ reporter gene.
-Galactosidase Activity in Liquid Cultures of
Yeast--
Cells were assayed for
-galactosidase activity by the
o-nitrophenyl-
-D-galactopyranoside (ONPG)
method (34).
-D-thiogalactopyranoside (1 mM)
for 3 h at 37 °C. Then, bacteria were harvested, resuspended in
35 ml of ice-cold PBS, and broken by sonication (five 30-s pulses at
100 watts, Sonics & Materials, Inc.). The resulting lysates were
centrifuged for 30 min at 4000 × g and used for
binding to amylose resin (New England Biolabs).
K14-T50
mutants, COS-7 cells (1 × 106) were transfected with
either 10 µg of wild-type pNL4.3 constructs or provirus harboring the
p7 H23C or p7
K14-T50 mutations using a standard calcium phosphate
method. 36 h after transfection, COS-7 cells were labeled with 200 µCi of [35S]methionine for 12 h.
Immunoprecipitation procedure was exactly the same as described above
for the HIV-1 Pr55gag (p6 L44P/F45S) mutant. A
ratio system previously used to assess levels of Vpr virion
incorporation relative to the levels found in the cell using other
virion proteins as standards (p66RT) was used (29). Densitometric
analysis of autoradiograms was performed with a Molecular Dynamics
personal densitometer using an ImageQuantTM software
version 3.22.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity unless it also contains a
prey vector that expresses an interacting protein as a fusion molecule
to the B42 acid blob transactivation domain (43). In the above
described system (37), the expression of the B42-fusion proteins is
conditional on the presence of galactose (Gal/Raff) in the culture
medium since the expression is directed by the GAL1 promoter.
His
Trp
medium. Five
independent transformants were then selected and streaked on either 1)
Glc Ura
His
Trp
X-Gal medium
or 2) Gal/Raff Ura
His
Trp
X-Gal medium. The yeast streaked on Glc Ura
His
Trp
X-Gal medium showed no indication
of
-galactosidase activity (by the observation of blue colonies),
indicating that no protein interaction occurs in the absence of
induction of the B42 fusions (data not shown). Fig.
1A shows the results from the
different transformation combinations when grown on Gal/Raff
Ura
His
Trp
X-Gal medium. As
previously shown by other groups (44, 45), our result confirmed that
the Pr55gag is capable of homo-oligomerization
(lane 3). Because Vpr has been shown to act as a
weak transactivator of some cellular promoters as well as the HIV-1
long terminal repeat (46), it was important to verify if Vpr could by
itself induce
-galactosidase expression when fused to the LexA DNA
binding domain. Lane 6 shows that in our yeast
two-hybrid system, Vpr does not seem to cause promoter transactivation
to levels appreciable for observation. However, when the
B42-Pr55gag fusion was introduced into the yeast
strain containing the LexA-Vpr fusion, strong
-galactosidase
expression was detected by the observation of blue colonies
(lane 7). This suggests that Vpr and the p55 Gag
precursor are directly interacting in vivo in the yeast cell
nucleus. Interestingly, the interaction between Vpr and
Pr55gag is observed only in one direction, that
is, when Vpr is fused to LexA sequences and the
Pr55gag to the B42 transactivation domain. When
the two fusions are switched, the interaction is not observed
(lane 4). In addition, evidence of Vpr
homo-oligomerization were observed using the yeast two-hybrid system
(lane 8), as previously shown by others (44).
When B42-Vpu (lanes 2 and 9), LexA
(lanes 5 and 10) or B42
(lanes 1 and 6) protein alone were
used, growth on X-Gal medium in the presence of galactose did not
result in the appearance of blue colonies. As well, controls in which
LexA-Vpu was introduced in yeast containing either B42-Vpr or
B42-Pr55gag did not show any sign of
-galactosidase expression (data not shown). These interaction
experiments were also confirmed using the ONPG colorimetric assay (Fig.
2B). The results presented in Fig. 1A suggest direct and specific binding of Vpr to the
Pr55gag.
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Fig. 1.
Specificity of interaction between Vpr and
the p55 Gag precursor in the yeast two-hybrid system.
A, the EGY48 reporter strain containing LexA, LexA-Vpr, or
LexA-Pr55gag was transformed with B42, B42-Vpu,
B42-Vpr, or B42-Pr55gag. The yeast cells (five
independent transformants) were then cultured on selective galactose
media containing X-Gal and assessed for their ability to interact
together. B, a clone shown to be positive for Vpr-Gag
interaction was grown in galactose selective media. Yeast cells were
lysed and immunoprecipitated with either rabbit anti-Vpr or mouse
anti-p24 sera. Samples were then electrophoresed on either a 8.5% (for
B42-Pr55gag) or 12.5% (for LexA-Vpr) SDS-PAGE,
transferred on nitrocellulose, and immunoblotted with either anti-Vpr
or anti-p24 antibodies.
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Fig. 2.
The p6 domain from the Pr55gag is
necessary and sufficient for the Vpr-Pr55gag interaction.
A, shown are the different constructs used to assay binding
in the two-hybrid system. MLVGag represents the full-length p65 Gag
precursor from the MLV, MLVGag/HIV-p6 represents the HIV-1 p6 domain
fused in-frame to the C terminus of the p65 Gag precursor from MLV. The
sequence of the nucleocapsid protein (NCp7) containing two zinc fingers
is shown. Amino acid substitutions were introduced in the NC domain by
site-directed mutagenesis. HIV-1 p7 H23C is a substitution of
His23 to Cys; HIV-1 p7 C28S/C49S contains substitutions of
Cys28 and Cys49 to Ser; HIV-1 p7 K14-T50 is
a deletion from Lys14 to Thr50 removing the two
zinc fingers. The amino acid sequence of p6 is shown. HIV-1
Pr55gag (p6 L44P/F45S) mutant is a double point
mutant in which Leu44 and Phe45 from the p6
domain were mutated to Pro and Ser, respectively. These mutations perturbed the highly conserved L-X-S-L-F-G
motif located in the C-terminal region of the p6 domain. The
L-X-S-L-F-G motif is underlined. L44P and F45S are indicated
in bold. MA, matrix (p17); CA, capsid
(p24); NC, nucleocapsid (p7). B, the interaction
strength between LexA-Vpr and B42-Pr55gag,
B42-MLVp65, B42-MLVp65/HIVp6, B42-HIV-1 Pr55gag
(p6 L44P/F45S), B42-HIV-1 Pr55gag (p7 H23C),
B42-HIV-1 Pr55gag (p7 C28S/C49S), or B42-HIV-1
Pr55gag (p7
K14-T50) was assessed using the
liquid
-Gal assay. The histogram represents averaged data from at
least three different experiments.
-galactosidase
activity in the yeast two-hybrid system using the ONPG colorimetric
assay. As shown in Fig. 2B, the
-galactosidase activity
detected for the
Pr55gag-Pr55gag
interaction is roughly 2 times the one detected for the association between Vpr and the Pr55gag. The
-galactosidase activity detected for the Vpr-MLV/HIVp6 interaction
is similar to the one observed for the
Vpr-Pr55gag interaction (Fig. 2B). As
expected, no interaction was observed with the wild-type p65 MLV Gag
precursor. Since the only difference between these two constructs is
the addition of the p6 domain, this result suggests that Vpr can
specifically associate with the p6 domain of HIV-1, and that
p6gag is necessary and sufficient for the
association with Vpr. In order to pinpoint the region of the p6 domain
that may act as a potential Vpr binding site, 2 out of the 52 amino
acids from the p6 domain were mutated in the
Pr55gag. Leu44 was changed for a Pro
and Phe45 for a Ser (Fig. 2A). These two
substitutions altered the conserved L-X-S-L-F-G motif of the
p6 domain shown to be important for Vpr incorporation (26, 27, 39). As
demonstrated in Fig. 2B, this mutation rendered the HIV-1
Pr55gag completely incapable of associating with
Vpr. Interestingly, this mutation in the context of a proviral
construct failed to incorporate Vpr (Fig. 5A). In order to
confirm that the lack of
-galactosidase activity detected with the
B42-MLVp65 and the B42-HIV-1 Pr55gag (p6
L44P/F45S) fusions was not due to stability problems or the absence of
protein expression, we performed Western blot analysis to detect these
fusion proteins. Western blot clearly demonstrated that the B42-MLVp65,
B42-MLV/HIVp6, and the B42-HIV-1 Pr55gag (p6
L44P/F45S) fusion proteins were expressed to similar levels in the
yeast EGY48 as determined by immunoprecipitation followed by Western
blot (data not shown). Together, these results suggest that the
mechanism by which Vpr is trans-incorporated into HIV-1 particles involves a direct physical interaction with the p6 domain of
the Pr55gag.
K14-T50 is a deletion of both zinc fingers) were fused in
translational frame with the B42 transactivator and assayed for
-galactosidase activity using the ONPG colorimetric assay in the
presence of LexA-Vpr (Fig. 2B). The results demonstrated that neither the HIV-1 Pr55gag (p7 H23C), the
HIV-1 Pr55gag (p7 C28S/C49S), nor the complete
deletion of the NCp7 zinc fingers (HIV-1 Pr55gag
(p7
K14-T50)) affected the binding between Vpr and the
Pr55gag. This result suggests that the integrity
of the NCp7 zinc fingers in the context of the
Pr55gag is not essential for the
Vpr-Pr55gag interaction.
-helix of Vpr and were shown
to be incapable of incorporation into virion particles (29). The Q65E
point mutation is located within the second helix of Vpr and affects its intranuclear localization (47). Finally, the SR79-80ID and the
R80A mutants do not lead to cell cycle arrest (11). Yeast containing
either wild-type LexA-Vpr or LexA-Vpr mutants were then transformed
with the following B42 fusions: the HIV-1
Pr55gag, the p65 MLV Gag precursor, the
MLVGag/HIV p6 fusion, and the HIV-1 Pr55gag (p6
L44P/F45S) double mutant, and assayed for interaction using the ONPG
colorimetric method. As shown on Fig. 3B, the E25K and the
A30F mutants were unable to interact with both the HIV-1
Pr55gag and the MLVGag/HIV p6 fusion, while the
other Vpr mutants were not affected. Similar expression of all LexA-Vpr
mutants was confirmed by Western blot analysis (data not shown).
Together, this observation suggests that the integrity of the predicted
N-terminal
-helical structure in Vpr is essential for the physical
association with the Pr55gag. As well, this
result confirms the relevance of the binding assay used in this study
since the two mutants shown not to interact with the
Pr55gag were shown not to incorporate virion
particles (29).
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Fig. 3.
The Vpr-Pr55gag interaction requires
an intact -helical structure in Vpr.
A, the amino acid sequence of the wild-type (WT)
Vpr is shown. The position of the predicted amphipathic
-helical
structures, the leucine/isoleucine-rich (LR) region, and the
basic amino acid-rich region in Vpr are also indicated. Design and
construction of the E25K, A30F, Q65E, SR79-80ID, and R80A Vpr mutants
were previously described (18, 29). B, the interaction
strength between B42-Pr55gag, B42-MLVGag,
B42-MLVGag/HIV-1 p6, and B42-HIV-1 Pr55gag (p6
L44P/F45S) double mutant and LexA-Vpr, LexA-VprE25K,
LexA-VprA30F, LexA-VprQ65E, LexA-VprSR79-80ID, or LexA-VprR80A
was assessed using the liquid
-Gal assay. The histogram represents
averaged data from at least three different experiments.
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Fig. 4.
In vitro interaction between Vpr
and the Pr55gag. A, equal amounts of MBP
(lane 3), MBP-Vpu (lane 2),
and MBP-Vpr (lane 1) were affinity-purified on
amylose resin and incubated with 5 µl of in vitro
translated [35S]methionine-labeled
Pr55gag. Following a 2-h incubation at 4 °C,
the samples were washed five times, eluted, loaded on a 12.5%
SDS-PAGE, and subjected to autoradiography. The input lane was loaded
with one-fifth of the amount of Pr55gag used in
the binding reactions (lane 4). B, to
test the strength of the Vpr-Gag interaction,
MBP-Vpr-Pr55gag bound complexes, were washed
four times with column buffer containing 200 mM NaCl, and
subsequently washed with 200, 300, 500, or 900 mM
NaCl-containing buffer. After these different washes, the different
samples were submitted to 10 mM maltose for elution of
MBP-bound complexes and analyzed on a 10% SDS-PAGE and by
autoradiography. C, equal quantity of MBP-Vpr fusion protein
affinity-purified by amylose resin were incubated for 3 h
(4 °C) with four different antibodies: a polyclonal rabbit serum
directed against recombinant Vpr (lane 2), a
rabbit serum generated against a peptide corresponding to the first 19 amino acids of Vpr (lane 3), anti-Vpu
(lane 4), anti-Myc (lane 5), and with
no antibody (lane 6). After incubation, unbound
and nonspecifically attached antibodies were washed twice. Then, 5 µl
of [35S]methionine-labeled, in vitro
translated Pr55gag were added to the different
samples, followed by a 2-h incubation at 4 °C. Samples were then
washed, eluted, and submitted to electrophoresis on a 12.5% SDS-PAGE
and to autoradiography.
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Fig. 5.
Virion incorporation of Vpr in wild-type and
mutant Gag HIV-1 proviruses. A, autoradiogram of a
radioimmunoprecipitation used to analyze the ability of Vpr to
incorporate into virion in wild-type and in HIV-1 p6 L44P/F45S mutant
in MT-4 cells. p6 LF-PS corresponds to the HIV-1 p6
L44P/F45S mutant proviral construct. Lanes 1-4
show the cell-associated proteins, while lanes
5-8 show the proteins from virions. Mock (lanes
1 and 5) and HIV-1 proviral construct mutated in
the Vpr initiation codon (R-) (lanes 2 and 6) transfected MT-4 cells were also included as
controls. R- proviral construct does not express Vpr. B,
autoradiogram of a radioimmunoprecipitation used to analyze the ability
of Vpr to incorporate into virions in wild-type and in the HIV-1 p7
H23C and HIV-1 p7 K14-T50 in COS-7 cells. Lanes
9-13 show virion-associated proteins. Mock (lane
9) and HIV-1 proviral construct mutated in the Vpr
initiation codon (R-) (lane 10) transfected COS-7
cells were also included as controls. C, relative virion
incorporation levels of Vpr in wild-type and Gag mutants based on
autoradiography scanning is depicted in this graph (see "Experimental
Procedures"). This quantification represents averaged data from two
independent experiments.
K14-T50 mutations. Because these viruses have been shown to
be highly affected in their ability to package HIV-1 RNA (40), these
viruses are not replication-competent. Consequently, we decided to use
COS-7 cells for viral particle production as described previously (23).
Using the same antisera as for the HIV-1 p6 L44P/F45S mutations, viral
proteins from lysed cells and sucrose cushion-pelleted virions were
immunoprecipitated for both the HIV-1 p7 H23C and the HIV-1 p7
K14-T50 proviral constructs. Fig. 5B shows the
virion-associated proteins from one of two independent experiments. As
can be seen, both virion particles generated from the HIV-1 p7
K14-T50 and the HIV-1 p7 H23C proviral constructs were still
competent in incorporating Vpr (Fig. 5B, lanes
11 and 12, respectively). Moreover,
quantification (Fig. 5C) using densitometric analysis
revealed that Vpr is trans-incorporated to levels similar to
wild-type virus into the HIV-1 p7 H23C mutant virions. Because the
HIV-1 p7
K14-T50 virion particles are highly affected in their Gag
processing (no detectable p66RT band, very low p24/p25 Gag; Fig.
5B, lane 11), we did not quantify Vpr
virion incorporation (Fig. 5C). Nonetheless, Fig.
5B (lane 11) shows that Vpr is still
incorporated in substantial amounts in the HIV-1 p7
K14-T50 virion particles.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical region located between amino acids 18 and 34 in the N terminus of Vpr is important for the incorporation of
this accessory protein (28-30, 50, 51). Our binding studies using five
Vpr mutants, E25K, A30F, Q65E, SR79-80ID, and R80A, showed that the
two mutants that were unable to interact with the
Pr55gag were the mutants that lost their ability
to be incorporated (29). This result also correlates the loss of
incorporation of Vpr to its incapacity to directly interact with the
Pr55gag. The observation that Vpr could only
interact with the Pr55gag when fused to the C
terminus of the LexA DNA binding domain (Fig. 1A,
lane 7) but not when fused to the B42
transactivator (Fig. 1A, lane 4)
suggests that this interaction requires proper conformation. It is
likely that either the B42-Vpr or the
LexA-Pr55gag fusion, or both, are not presented
in the proper structure to expose their respective binding domain.
Interestingly, we suspect that the p6-binding motif of Vpr requires
more than just the predicted N-terminal
-helical domain since
attempts to compete the in vitro Vpr-Pr55gag association with a series of Vpr
peptides were unsuccessful. Peptides harboring Vpr amino acids 1-19,
19-35, or 23-37 could not compete the interaction between Vpr and the
Pr55gag. Moreover, Yao et
al.2 demonstrated that
fusion proteins containing amino acids 1-62, which harbor the
predicted N-terminal
-helical moiety of Vpr, are incapable of
incorporating virion particles, while polypeptides fused to amino acids
1-80 of Vpr are efficiently incorporated. These results suggest that
the presence of the leucine/isoleucine-rich domain of Vpr (amino acids
60-80) might be important for correct folding or exposition of the
predicted N-terminal
-helical region. Similar results have also been
observed by Sato et al. (52). It is noteworthy that our
anti-Vpr peptide serum directed against the N-terminal region of Vpr
(amino acids 1-19) was more effective in affecting the interaction
with the Pr55gag than the antibody directed
against recombinant Vpr (Fig. 4C), which principally
recognizes epitopes lying between amino acids 19 and
72.3 This suggests that, even
though the leucine/isoleucine-rich region of Vpr might be important,
the critical region for the Vpr-Pr55gag
interaction is the predicted N-terminal
-helical structure of Vpr.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Roger Brent for the generous gift of plasmids and yeast strains used in the two-hybrid system. We also thank Dr. Erica Golemis for helpful advice that enabled this work to be done and Nash Daniel and Drs. Béatrice Allain and Andrew J. Mouland for fruitful discussion.
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Addendum |
---|
During review of this work, a paper appeared (Selig, L., Pages, J.-C., Tanchou, V., Prévéral, S., Berlioz-Torrent, C., Liu, L. X., Erdtmann, L., Darlix, J.-L., Beuarous, R., and Benichou, S. (1999) J. Virol. 73, 592-600) in which a direct interaction between Vpr and the Pr55gag of HIV-1 was reported.
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FOOTNOTES |
---|
* This work was supported in part by grants from the Medical Research Council (MRC) of Canada and Theratechnologies Inc. (to E. A. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a studentship from the MRC of Canada.
§ Recipient of a MRC scientist career award. To whom correspondence should be addressed. Tel.: 514-343-5967; Fax: 514-343-5995; E-mail: Eric.cohen{at}umontreal.ca.
3 C. Lavallée and E. A. Cohen, unpublished observations.
2 X.-J. Yao, G. Kobinger, S. Dandache, N. Rougeau, and E. A. Cohen, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are:
HIV-1, human
immunodeficiency virus type 1;
HIV-2, human immunodeficiency virus type
2;
SIV, simian immunodeficiency virus;
MLV, Moloney murine leukemia
virus;
ONPG, o-nitrophenyl--D-galactopyranoside;
MBP, maltose-binding protein;
Glc, glucose;
Gal/Raff, galactose and
raffinose;
RT, reverse transcriptase;
PCR, polymerase chain reaction;
PBS, phosphate-buffered saline;
X-Gal, 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside.
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REFERENCES |
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