The Multimerization of Human Immunodeficiency Virus Type
I Vif Protein
A REQUIREMENT FOR Vif FUNCTION IN THE VIRAL LIFE CYCLE*
Shicheng
Yang,
Yong
Sun, and
Hui
Zhang
From The Dorrance H. Hamilton Laboratories, Center for Human
Virology, Division of Infectious Diseases, Department of Medicine,
Jefferson Medical College, Thomas Jefferson University,
Philadelphia, Pennsylvania, 19107
Received for publication, June 6, 2000, and in revised form, August 16, 2000
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ABSTRACT |
The Vif (virion
infectivity factor protein of human
immunodeficiency virus type I (HIV-1) is essential for viral
replication in vivo and productive infection of peripheral
blood mononuclear cells, macrophages, and H9 T-cells. However,
the molecular mechanism(s) of Vif remains unknown and needs to be
further determined. In this report, we show that, like many other
proteins encoded by HIV-1, Vif proteins possess a strong tendency
toward self-association. In relatively native conditions, Vif proteins
formed multimers in vitro, including dimers, trimers, or
tetramers. Through in vivo binding assays such as
coimmunoprecipitation and the mammalian two-hybrid system, we also
demonstrated that Vif proteins could interact with each other within a
cell, indicating that the multimerization of Vif proteins is not simply
due to fortuitous aggregation. Further studies indicated that the
domain affecting Vif self-association is located at the C terminus of
this protein, especially the proline-enriched 151-164 region.
Moreover, we found that a Vif mutant with deletion at amino acid
151-164 was unable to rescue the infectivity of vif-defective viruses generated from H9 T-cells, suggesting
that the multimerization of Vif proteins could be important for Vif function in the viral life cycle. Our studies identified a new feature
of Vif and should accelerate our understanding of its role in HIV-1 pathogenesis.
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INTRODUCTION |
The accessory genes of
HIV-1,1 including
vif, vpr, nef, and vpu,
have been shown to play important roles during HIV-1 infection (1). It
has been demonstrated that Vif affects the late stages of the viral
life cycle, possibly through the assembly of viral particles (2-4).
The vif-defective (vif
) viruses are able to penetrate into target cells but not accomplish reverse transcription (4-7). The requirement for Vif, however, is cell type-specific. The
vif
viruses exhibit a negative phenotype only when
produced from primary T-lymphocytes, terminally differentiated
macrophages, or a few T-lymphoid cell lines, such as H9. These cells
were entitled as "nonpermissive" cells. In some T-cell lines such
as SupT1, C8166, and other non-T-cells such as HelaCD4 cells, however,
productive replication of vif
HIV-1 viruses can be
achieved. These cell lines therefore were named as "permissive"
cells (2, 4, 8). There are two possibilities for Vif function in the
nonpermissive cells; Vif may counteract an endogenous inhibitor
existing in the nonpermissive cells or alternatively, substitute a Vif
homologue that exists in the permissive cells but not nonpermissive
cells (9). A recent study showed that the permissive HelaCD4 cells expressing the HIV-1F12 Vif were resistant to the
replication of wild-type HIV-1, suggesting that there may be a Vif
homologue in the permissive cells that was inhibited by
HIV-1F12 Vif (10). Conversely, the progeny viruses
generated from the heterokayons that were formed between permissive and
nonpermissive cells showed a phenotype similar to that generated from
the nonpermissive cells. This result suggested that nonpermissive
cells, most likely the natural targets of HIV-1, contain a potent
endogenous inhibitor of HIV-1 replication that is counteracted by Vif
(11, 12). However, the nature of endogenous inhibitor and the molecular mechanism(s) regarding how Vif interacts with it remain unknown.
Recently, it has been shown that Vif is associated with a complex in
the virus-producing cells (13). Although it has been demonstrated that
Vif of HIV-1 interacts with the NCp7 domain of p55 Gag precursor
in vitro through its positively charged amino acid-enriched
C terminus and colocalizes with Gag precursors in a cell, no direct
interaction was observed between Vif and Gag precursors (13-16). We
further demonstrated that Vif is an RNA binding protein and able to
form an RNase-sensitive messenger ribonucleoprotein complex with viral
unspliced RNA in the cytoplasm of HIV-1-infected cells. As Vif-RNA
binding could be displaced by Gag-RNA binding, Vif may mediate viral
RNA engagement with HIV-1 Gag precursors and thus could be involved in
genomic RNA folding and packaging (31). In this study, we demonstrate a new biochemical characteristic of Vif protein; Vif proteins have a
strong tendency to form multimers, which could play an important role
for the Vif function in HIV-1 life cycle.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructions--
With infectious clone pNL4-3 as
template, deletion mutants of HIV-1 Vif were generated by polymerase
chain reaction (PCR)-mediated and site-directed mutagenesis (17). The
PCR-generated wild-type vif gene and its mutants were then
inserted into pCITE-4a vector (Novagen, Madison, WI) for in
vitro translation. The vif gene was also inserted into
pGEX vector for in vitro expression and isolation of GST-Vif
fusion protein. For studying intracellular Vif-Vif interaction,
vif genes were tagged with FLAG (DYKDDDDK) or c-Myc
(EQKLISEEDL) epitope-encoding sequences at the 3' terminus, respectively, via PCR. These tagged vif genes were then
inserted into the vector pCI-Neo, which contains a chimeric intron just downstream of the cytomegalovirus enhancer and immediate early promoter
(Promega, Madison, WI). The resulting plasmids were named pCI-vif-c-Myc
or pCI-vif-FLAG, respectively. For mammalian two-hybrid analysis,
pGal-Vif or pGal-Vif
151-164 were constructed by replacing the
HindIII-BamHI fragment (containing vp
gene) of pSG5GalVP with a PCR-amplified complete vif gene or
its mutant
151-164. The pVif-VP or pVif
151-164-VP were
constructed by replacing the EcoRI-BglII fragment
(containing gal4 gene) of pSG5GalVP with a PCR-amplified complete vif gene or its mutant
151-164, respectively
(18). The integrity of all the constructs was confirmed by DNA sequencing.
Protein Expression and in Vitro Binding Assays--
The vector
pGEX, with or without the vif gene, was transformed into
BL21 competent cells (Novagen, Madison, WI). After growth at 37 °C
to ~0.6 optical density, The expression of GST or GST-Vif proteins were induced by 0.4 mM
isopropylthio-
-D-galactoside. The bacterial cells were
lysed by adding lysing buffer (1% Triton X-100, 0.1 mg/ml lysozyme, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 1 µg/ml aprotinin), followed by sonication. The
sample was pelleted at 12,000 × g for 10 min at
4 °C, and the supernatant was applied to a glutathione-conjugated
agarose bead (Sigma) column. After batch binding, the matrix was
washed by the addition of 10 bed volumes of phosphate-buffered saline 3 times. The GST or GST-Vif-conjugated agarose beads were then aliquoted
and stored at
20 °C. Conversely, 35S-labeled Vif or
its mutant proteins were synthesized utilizing SPT3 kits (Novagen,
Madison, WI). The protocol supplied by manufacturer was followed. After
in vitro translation, RNase A (0.2 mg/ml) was added to stop
the reaction and remove tRNAs and the in vitro transcribed-mRNA. The trichloroacetic acid-insoluble radioactive amino acids were quantitated in the presence of a scintillantion mixture.
For GST pull-down assays, a GST- or GST-Vif-conjugated bead slurry was
mixed with 35S-labeled Vif or its mutants (50,000 cpm) in a
binding buffer (150 mM NaCl, 20 mM Tris-HCl (pH
7.5), 0.1% Triton X-100). After binding at 4 °C for 1 h, the
mixture were centrifuged at 3,000 × g for 1 min, and
the beads were washed with binding buffer three times. The
35S-labeled Vif proteins were dissociated from beads by
adding SDS-containing loading buffer, and heating at 95 °C for 5 min. The samples were then electrophoresed in SDS-PAGE gels (15%
Tris-HCl ready gel made by Bio-Rad, Hercules, CA). After
treatment with the fixing buffer (10% acetic acid, 10% methanol) and
then Amplify (Amersham Pharmacia Biotech), the gels were dried
and exposed to x-ray film or quantitatively analyzed utilizing a
PhosphorImager (Molecular Dynamics, Sunnyview, CA).
Furthermore, in vitro-translated, 35S-labeled
Vif (50,000 cpm) was also directly loaded into a 4-20% Tris/glycine
gel (SDS-free) via 10% glycerol-containing loading buffer, with SDS at
various concentrations, and electrophoresed with an SDS-free
Tris/glycine running buffer. After fixing and drying, the gel was
directly subjected to autoradiography.
Western Blotting and Coimmunoprecipitation--
The COS-1 or
293T cells were transfected with 5 µg of pCI-vif-c-Myc and
pCI-vif-FLAG using a calcium phosphate precipitation method (17, 19).
After 48 h, the cells were lysed in a cell lysing buffer (150 mM NaCl, 50 mM Tris-HCl, (pH 8.0), 5 mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml
leupeptin, 2 µg/ml pepstatin A). For direct Western blotting, the
whole-cell lysates were mixed with acetone (1:3). The mixture was
incubated on ice for 20 min, followed by centrifugation at 12,000 × g for 10 min. The pellets were air-dried and resuspended
in SDS-containing sample buffer. The samples were electrophoresed in
SDS-PAGE gels and then electronically transferred onto a
nylon/nitrocellulose membrane. The primary antibodies, goat anti-c-Myc
antibody (A14) (Research Antibodies, Santa Cruz, CA), or mouse
anti-FLAG antibody (M2) (Stratagene, La Jolla, CA) were used to bind
the samples, respectively. The horseradish peroxidase-conjugated anti-goat IgG antibody or anti-mouse IgG antibody (Research Antibodies, Santa Cruz, CA) was used as the secondary antibody. A
Chemiluminescence-based system (ECL; Amersham Pharmacia
Biotech) was used to visualize the antigen-antibody binding.
For coimmunoprecipitation, cell lysates from COS-1 or 293T cells
expressing Vif-FLAG and/or Vif-c-Myc were incubated with A14 anti-c-Myc
antibody (Santa Cruz) (1 µg/ml) by mixing 12 h at 4 °C,
followed by incubation with protein A-conjugated Sepharose CL-4B
(Amersham Pharmacia Biotech) for an additional 2 h. The pellet was
washed three times with cell lysing buffer. The pellet was then
resuspended in SDS-containing buffer, heated at 95 °C, and
centrifuged at 12,000 × g. The supernatant was then
subjected to SDS-PAGE. After transfer onto a nylon/nitrocellulose
membrane, the samples were detected with a mouse M2 anti-FLAG antibody. An horseradish peroxidase-conjugated anti-mouse IgG (Research Antibodies, Santa Cruz, CA) was used as a secondary antibody.
Mammalian Two-hybrid System Assay--
A mammalian two hybrid
system, which was modified from the GAL4-based yeast two-hybrid assay,
was used to study the self-association of HIV-1 Vif proteins in
vivo (18, 20). The procedure was as described previously, with
some modifications (18, 20). Briefly, 5 µg of pGal-Vif and pVif-VP
were cotransfected with pG5BCAT into COS-1, using the Superfect
transfection reagent (Qiagen, Valencia, CA). 48 h
post-transfection, the cells were lysed in reporter lysing buffer
(Promega, Madison, WI) and subjected to a chloramphenicol
acetyltransferase (CAT) assay, as described previously (19).
Single-round Viral Infectivity Assays--
The biological
activity of Vif mutants was evaluated by using a single-round viral
infectivity assay, with some modifications (7). To generate recombinant
HIV-1 viruses, H9 cells were transfected with 5 µg of
pNL4-3
vif
env, pMD.G (containing vesicular
stomatitis virus (VSV) envelope), and wild-type vif gene or
its mutants (in pCI-Neo construct) by electroporation (7, 21). The
electroporation (350 V, 250 microfarad, 5.1-6.3 ms) was performed by a
gene pulser apparatus and capacitance (Bio-Rad, Hercules, CA).
Thereafter, conditioned medium (RPMI 1640 plus 10% fetal bovine serum)
was used to maintain the transfected H9 cells. Two days after
transfection, the viral particles in supernatant were collected and
pelleted via ultracentrifugation (7). After normalization by the
HIV-1 p24 antigen level, which was detected via enzyme-linked
immunosorbent assays (kits from DuPont), the viruses were used to
infect 5 × 105 HeLaCD4-CAT cells (22). 48 h
post-infection, the cells were lysed in reporter lysing buffer
(Promega) and subjected to CAT assays.
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RESULTS |
Vif Proteins Can Form Multimers in Vitro--
To examine whether
Vif proteins have a tendency toward self-association, GST-Vif was
expressed in BL 21 bacterial cells and isolated onto
glutathione-conjugated agarose beads. In vitro-translated, 35S-labeled Vif proteins were allowed to incubate with the
GST-Vif-conjugated beads. The bead-associated 35S-labled
Vif was then analyzed by SDS-PAGE, followed by direct autoradiography.
Fig. 1A illustrates that
GST-Vif (lane 2), but not GST (lane 3), can
strongly bind to 35S-labeled, in
vitro-translated Vif protein, indicating a Vif-Vif interaction.
Further, 35S-labeled, in vitro-translated HIV-1
Vif protein was directly loaded onto a Tris/glycine-native gel
(SDS-free) for electrophoresis, with loading buffers containing 10%
glycerol only or SDS at various concentrations. At the native or
relatively native conditions, the 35S-labeled Vif proteins
migrated in the 4-15% Tris/glycine gels as monomers (23 kD), dimers
(46 kD), and trimers (69 kD) or tetramers (92 kD) (Fig. 1B).
With the increment of concentrations of SDS in the loading buffer, the
major forms of Vif eventually became a monomer (23 kD). When the sample
was heated at 95 °C for 5 min, all the multimers of Vif proteins
disappeared, suggesting that the Vif-Vif binding is unlikely to be
covalent. It is notable that, prior to the sample loading,
35S-labeled, in vitro-translated HIV-1 Vif
protein was treated with RNase A to remove possible RNA contamination.
Therefore, the Vif-Vif binding should be RNA-independent.

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Fig. 1.
Vif self-association in a cell-free
system. A, in vitro translated,
35S-labeled HIV-1NL4-3 Vif proteins were
allowed to bind with GST-Vif conjugated on beads. After binding, the
bead-associated 35S-labeled Vif was analyzed via SDS-PAGE
and direct autoradiography. B, Vif proteins form dimers and
multimers in native or mild-denatured loading buffer. In
vitro-translated 35S-labeled HIV-1NL4-3
Vif proteins were loaded directly onto a 4-20% Tris-HCl gel
(SDS-free) with native loading buffer (62.5 mM Tris-HCl (pH
6.8), 20% glycerol) plus SDS at different concentrations.
Electrophoresis was performed with a Tris/glycine running buffer
containing 0.05% SDS, followed by autoradiography. ME,
-mercaptoethanol.
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The Binding Site for Vif Multimerization Is Located in the C
Terminus--
To determine the binding sites for Vif multimerization,
a series of deletions in Vif protein have been generated through
PCR-based mutagenesis, followed by in vitro translation in
the presence of [35S]methionine. These Vif mutants were
then allowed to bind to GST-Vif fusion protein conjugated to agarose
beads. Fig. 2A indicates that
the deletion of the C terminus in Vif protein severely loses the
Vif-Vif binding activity. Further studies indicated that deletion at
amino acid 151-164 would significantly decrease this binding ability
(Fig. 2A). This result was further confirmed by native multimer formation assay. In the presence of 0.1% SDS, Vif mutants
151-192 and
151-164 were unable to form multimers, whereas
other mutants were able to do so (Fig. 2B). It is notable
that there are several positively charged amino acids in the 151-164
fragment. The mutants that substitute these positively charged amino
acids, generated by Goncalves et al. (23), have been
examined for this Vif-Vif binding. However, all these mutants still
contain Vif-Vif binding ability (data not shown). It is also notable
that there are several prolines (Pro156,
Pro161, Pro162, and Pro164)
in this fragment. Among these prolines, Pro161 is highly
conserved in various strains of HIV-1 or simian immunodeficiency virus. Whether these prolines are important for Vif-Vif binding remains to be further verified.

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Fig. 2.
The effect of Vif mutants on Vif-Vif
interactions. A, a series of deletions along the Vif
192 amino acids were generated via PCR-based mutagenesis and in
vitro translation. The in vitro-translated,
35S-labeled HIV-1NL4-3 Vif protein and its
mutants were allowed to bind to GST-Vif conjugated on agarose beads.
The bead-associated, 35S-labeled Vif protein and its
mutants were subjected to SDS-PAGE and visualized by direct
autoradiography. The values were obtained by quantitation with
densitometry of the autoradiographs. The ratio of bound Vif
versus the input was then calculated. The ratio of
GST-Vif-bound 35S-labeled wild-type Vif and
35S-labeled wild-type Vif input was further set as 100%
(with the standard deviations). The relative binding ability of Vif
mutants was thus determined. In most cases, the data reflect at least
five independent experiments. WT, wild-type. B, in
vitro-translated 35S-labeled HIV-1NL4-3
Vif protein and its mutants (50,000 cpm count for each) were loaded
directly onto a 4-20% Tris-HCl gel (SDS-free), with loading buffer
(62.5 mM Tris-HCl (pH 6.8), 20% glycerol) plus 0.1% SDS.
Electrophoresis was performed with a Tris/glycine running buffer
containing 0.05% SDS, followed by autoradiography.
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Vif-Vif Interactions within a Cell--
To elucidate the
possibility that Vif self-association also occurs intracellularly, we
utilized a coimmunoprecipitation method. The Vif protein was tagged
with c-Myc or FLAG epitope at its C terminus, respectively, and
expressed in the COS-1 cells. Fig. 3
indicated that the expression of c-Myc-tagged Vif and FLAG-tagged Vif
could be detected via Western blotting, with mouse anti-c-Myc epitope
antibody or goat anti-FLAG epitope antibody, respectively (top
two panels). To study Vif-Vif interaction, the cell lysates were
immunoprecipitated with anti-Myc antibody and then subjected to
SDS-PAGE, followed by Western blotting. The goat anti-FLAG antibody was
used to detect FLAG-tagged Vif. Fig. 3 demonstrated that the
FLAG-tagged Vif was coprecipitated with Myc-tagged Vif when mouse
anti-Myc antibody was utilized for the immunoprecipitation, suggesting
a Vif-Vif interaction within a cell (Fig. 3, bottom panel).

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Fig. 3.
Coimmunoprecipitation method to study Vif-Vif
interactions within cells. COS-1 cells were transfected with
vectors harboring FLAG or c-Myc tagged Vif. After 54 h of
incubation at 5% CO2, 37 °C, 20 µg of total cell
lysates were resolved by 15% Tris-HCl gel. The Vif proteins were
detected by Western blotting (WB) using an M2 anti-FLAG
monoclonal antibody and A14 anti-c-Myc polyclonal antibody,
respectively. For coimmunoprecipitation, the whole-cell lysates from
the same batch were subjected to immunoprecipitation (IP)
with A14 anti-c-Myc polyclonal antibody. Immunoprecipitates were
resolved at 15% Tris-HCl gel, transferred onto a membrane, and then
detected using an M2 anti-FLAG antibody.
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Alternatively, the in vivo Vif-Vif interaction was examined
by the mammalian two-hybrid system. A fusion protein composed of VP16
and Gal4 is able to activate the Gal4 response element-contained E1b
promoter. Gal4 would function as a DNA binding domain, whereas VP16
will function as a DNA activation domain. HIV-1 Vif protein was allowed
to replace VP16 or Gal4 domain, respectively (Fig. 4A). If the interaction
between Vif proteins take place, the VP16 and Gal4 domain would be
brought together, and the Gal4 binding sequence-contained E1b promoter
would be activated. Fig. 4B indicated that, like Rev-Rev
interactions, Vif in Vif-VP16 fusion protein could bind to Vif in the
Gal4-Vif fusion protein and activate the expression of CAT (lane
6). As controls, pGal-Vif or pVif-VP alone were unable to activate
CAT expression (lanes 3 and 4). Fig.
4B also shows that Vif mutant
151-164, which did not
have the ability to interact with Vif protein in other systems, also could not interact with Vif in this system (lane 7).

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Fig. 4.
Mammalian two-hybrid system to study Vif-Vif
interaction. A, a schematic map showing the plasmids
utilized in the experiments. B, COS-1 cells were transfected
with plasmids combined with various vectors. After 48 h, cell
lysates were harvested and subjected to CAT analyses.
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Deletion of the Vif-Vif Binding Domain Severely Decreases the Vif
Function in the Viral Life Cycle--
As mentioned previously, Vif
functions in the late events of HIV-1 life cycle and is required by
nonpermissive cells, such as peripheral blood mononuclear cells,
macrophages, and H9 T-cells (2-4). To investigate the physiological
significance of Vif multimerization, we examined whether Vif mutant
(
151-164), which is unable to form multimers in the cell-free
system and within cells, is able to complement Vif function in the
viral life cycle. To this end, a single-round viral infectivity assay
was adapted. Wild-type Vif or its mutants were expressed in the
nonpermissive H9 T-cells. At the same time, pseudotyped (with VSV
envelope) HIV-1 viruses, without vif and env in
their genome, were generated from these cells. After
ultracentrifugation for enrichment, the recombinant viruses were
allowed to infect the target cells (HelaCD4-CAT), which harbor an
expression cassette containing the HIV-1 long terminal repeat
promoter-driven CAT gene. The viral infectivity was measured by the
level of CAT gene expression in the target cells, which is driven by
the HIV-1 Tat protein expressed by the newly synthesized proviruses.
Fig. 5 demonstrates that, when the wild-type vif gene was expressed in the
vif-defective HIV-1 virus-producing nonpermissive H9
T-cells, the viral infectivity could reach a high level (lane
2). However, when Vif
151-164 was expressed in the
vif-defective HIV-1 virus-producing nonpermissive H9
T-cells, the viral infectivity was almost unaltered (lane
3), compared with the vif-defective HIV-1 viruses
(lane 4). These data indicated that the 151-164 deletion
severely decreased the function of Vif protein and made it unable to
rescue the infectivity of the vif-defective HIV-1 viruses
generated from nonpermissive T-cells. It is notable that another group
also demonstrated that this fragment is essential for Vif function
(24). This experiment demonstrated that multimerization of Vif proteins
is required for Vif function.

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Fig. 5.
Viral infectivity affected by Vif or Vif
mutants. The pCI-Neo constructs, containing wild-type
vif gene or its mutants,
pNL4-3 env vif plasmid and pMD.G (containing
VSV envelope (env)), were cotransfected into H9 cells to
generate the pseudotyped viral particles. After concentration via
ultracentrifugation, the viral particles were normalized by HIV-1 p24
antigen. In the presence of polybrene (8 µg/ml), the viruses were
used to infect HelaCD4-CAT cells. After 48 h, the cell lysates
were collected and subjected to CAT analyses. Lane 1,
pNL4-3; lane 2, pNL4-3 env vif,
VSV env plus wild-type vif; lane 3,
pNL4-3 env vif, VSV env plus
vif 151-164; lane 4,
pNL4-3 env vif, VSV env plus
vif 144-150; lane 5,
pNL4-3 env vif, VSV env plus
pCI-Neo vector only. The value of wild-type vif
complementation was set as 100%. The relative values of the other
samples were calculated accordingly. This figure is representative of
three independent experiments. Values are means ± standard
deviations.
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DISCUSSION |
Many HIV-1 proteins, including Gag, protease, reverse
transcriptase, integrase, glycoprotein 41 (gp41), Tat, Rev, Vpr, and Nef, have been shown to form dimers or multimers in vitro
and in vivo. The formation of dimers or multimers has been
demonstrated to be important for their functions in the lentiviral life
cycle (25-28). In addition, multimerization is critical to the
biological activity of many prokaryotic and eukaryotic proteins and is
a common mechanism for the functional activation/inactivation of proteins. In this study, we analyzed the multimerization potential of
HIV-1 Vif proteins via various complementary methods. The in vitro-translated, 35S-lableled Vif proteins were able
to form multimers in the native environment. Conversely, GST-Vif fusion
proteins, rather than GST proteins, which were generated from the
bacterial expression system, were able to bind to the in
vitro-translated, 35S-lableled Vif proteins. Further,
coimmunoprecipitation and a mammalian two hybrid system also
demonstrated a Vif-Vif interaction intracellularly. These in
vitro and in vivo data strongly support the notion that
Vif proteins are able to form multimers. As the deletion of the domain
that is essential for the Vif-Vif binding severely decreases the
function of Vif in the nonpermissive cells, multimerization of Vif
could be important for its function in the HIV-1 life cycle. However,
as the function of Vif protein in the life cycle remains largely
unknown, the precise role of Vif multimerization and the active form(s)
(i.e. monomer, dimer, or tetramer) of Vif protein
in the virus-producing cells remains to be determined.
The domain for Vif multimerization has been located in a positively
charged amino acid- and proline-enriched fragment (amino acid 151-164)
(Fig. 2). As the positively charged amino acids in this region are not
responsible for the Vif-Vif interaction, whether the prolines are
important remains to be clarified. It is notable that a highly
conserved motif, SLQYLAL (amino acid 144-150 for
HIV-1NL4-3), is close to this domain. It has also been
shown that Ser165 is phosphorylated by the
mitogen-activated protein kinase (p44/42) of Vif, and this
phosphorylation is important for Vif function (30). As these residues
are close to the domain for multimerization, it is possible that the
multimerization of Vif proteins is regulated by phosphorylation in the
virus-producing cells. Interestingly, the positively charged amino
acids (replaced in B4 and B7 mutants) in the C terminus of Vif are
responsible for Vif-NCp7 binding in vitro (14). Recently, we
demonstrated that HIV-1 Vif is an RNA binding protein and an integral
component of a messenger ribonucleoprotein complex of viral RNA in the
cytoplasm and could be involved in the viral RNA packaging process
(31). In contrast to interactions with NCp7 via its C terminus, Vif
binds to RNA via its N terminus. Although more RNA would bind to Vif
than to Gag at the same conditions when RNA is mixed with Vif or Gag
separately, RNA will only bind to Gag but not Vif when Vif protein is
mixed together with RNA and NCp7 (31). This "displacement" could be
because of various mechanisms and is under investigation. However, as
the domains for Vif multimerization and for Vif-NCp7 binding are quite
close in location or possibly overlap, it is possible that the
interaction between Vif and Gag, as well as the interactions among Vif,
RNA, and Gag, is regulated by Vif multimerization.
Thus, the finding of Vif multimerization may be helpful in
understanding the structure-function relationship of Vif protein, identifying the molecular mechanism(s) of HIV-1 Vif in the viral life
cycle. In addition, these data provide a promising intervention target
for anti-HIV-1 agent development.
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ACKNOWLEDGEMENTS |
We thank Drs. Roger J. Pomerantz,
Geethanjali Dornadula, Charvi A. Patel, and Jianhua Fang
for critical review of the manuscript and valuable discussions. We also
thank Dr. Dana Gabuzda for providing plasmids of Vif C-terminal
mutants (B1-B7).
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FOOTNOTES |
*
This work was supported by Thomas Jefferson University funds
and the Margaret Q. Landenberger Research Foundation (to H. Z.).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.
To whom correspondence should be addressed: The Dorrance H. Hamilton Laboratories, Center for Human Virology, Division of Infectious Diseases, Dept. of Medicine, Jefferson Medical College, Thomas Jefferson University, 1020 Locust St., Suite 329, Philadelphia, PA 19107. Tel.: 215-503-0163; Fax: 215-923-1956; E-mail:
Hui.Zhang@ mail.tju.edu.
Published, JBC Papers in Press, November 8, 2000, DOI 10.1074/jbc.M004895200
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ABBREVIATIONS |
The abbreviations used are:
HIV-1, human
immunodeficiency virus type I;
GST, glutathione
S-transferase;
CAT, chloramphenicol acetyltransferase;
PCR, polymerase chain reaction;
PAGE, polyacrylamide gel electrophoresis;
VSV, vesicular stomatitis virus.
 |
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