§ INSERM Unit 372, Université de la Méditerranée, 163 Avenue de Luminy, 13276 Marseilles Cedex 09, France, ¶ INSERM Unit 119, Institut Paoli-Calmettes, Université de la Méditerranée, 27 Boulevard Lei Roure, 13009 Marseilles, France, and §§ LCPMI, Free University of Brussels, Boulevard du Triomphe, 1050 Brussels, Belgium
Received for publication, October 4, 2000, and in revised form, February 8, 2001
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ABSTRACT |
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The virus infectivity factor (Vif) protein
facilitates the replication of human immunodeficiency virus type 1 (HIV-1) in primary lymphocytes and macrophages. Its action is strongly
dependent on the cellular environment, and it has been proposed that
the Vif protein counteracts cellular activities that would otherwise limit HIV-1 replication. Using a glutathione S-transferase
pull-down assay, we identified that Vif binds specifically to the Src
homology 3 domain of Hck, a tyrosine kinase from the Src family. The
interaction between Vif and the full-length Hck was further assessed by
co-precipitation assays in vitro and in human cells. The
Vif protein repressed the kinase activity of Hck and was not itself a
substrate for Hck phosphorylation. Within one single replication cycle
of HIV-1, Hck was able to inhibit the production and the infectivity of vif-deleted virus but not that of wild-type virus.
Accordingly, HIV-1 vif The Vif protein of human immunodeficiency virus type 1 (HIV-1)1 is required for
efficient virus replication in peripheral blood T lymphocytes in
vitro (1-3), since vif-deleted (vif
Human cell lines are restrictive or permissive based on their ability
to support the replication of HIV-1 in the absence of Vif. Restrictive
human T cell lines H9 and HUT78 are resistant to infection by HIV-1 in
the absence of Vif (1, 9, 10). In contrast, permissive cell lines such
as Jurkat, C8166, HeLa, and 293 produce high amounts of vif
The Vif protein acts late in the virus life cycle, and, in its absence,
virus particles exhibit an abnormal condensed core as seen by electron
microscopy (13-15). The co-localization of Vif with the major
component of the viral core, i.e. Gag (16), and the direct
association of Vif with Gag in vitro and in infected cells (17) support
the hypothesis that Vif acts as a scaffold protein regulating the
virion morphogenesis. Interestingly, it was recently shown using highly
purified HIV-1 virus that Vif is not significantly incorporated into
the HIV-1 budding virions (18). This finding implicated that, during
virus budding, Gag and Gag-Pro-Pol precursors are incorporated into
newly synthesized virus particles where maturation process of
structural proteins may occur, whereas Vif remains associated to the
cell membrane. Consequently, cellular factors have been suggested to
play a crucial role in the retention of Vif at the plasma membrane of
the infected cells (17, 18).
Vif of HIV-1 contains a proline-rich motif (PPLP) localized within its
C-terminal domain, which is highly conserved among the different
subtypes of HIV-1 (Fig. 1). Src homology 3 (SH3) domains correspond to
intracellular modules, which mediate interaction with proteins
containing proline-rich motifs. The presence of SH3 domains was
reported in a wide variety of proteins participating to signal
transduction pathways, such as tyrosine kinases and adaptor proteins.
In this work, we hypothesize that a direct interaction may occur
between Vif and SH3 domains. We show that Vif of HIV-1 binds
preferentially to the SH3 domain of the tyrosine kinase Hck and not
significantly to other SH3 domains representative of the tyrosine
kinases families and of adaptor proteins. The interaction of Vif with
the full-length Hck being confirmed, we have analyzed the consequence
of this interaction on vif Cells
Human kidney 293 cells were maintained in Dulbecco's modified
Eagle's medium (supplemented with 10% fetal calf serum, antibiotics (penicillin/streptomycin, 100 µg/ml), and 2 mM glutamine.
U937 promonocytes and SupT1, Jurkat JH6.2, C8166, and H9
CD4+ T lymphocytes were grown in 1640 RPMI medium
supplemented with 10% fetal calf serum, antibiotics, and 2 mM glutamine. For Jurkat cells stably expressing Hck, G418
was added at 2 mg/ml. Human CD4+ T lymphocytes and
monocytes/macrophages were isolated from peripheral blood mononuclear
cells as described (19).
DNA Constructions
Vif Vectors--
pGST-Vif and Pos7-Vif, strain NL4.3 of HIV-1,
were described previously (17). GST-Vif was also cloned in the Pos7
vaccinia virus expression vector by PCR to generate Pos7-GST-Vif. The
GST-Vif DNA was amplified by PCR using the primers
GST-Vif/NcoI (s), and GST-Vif/XhoI (as), and
inserted in the Pos7 vector by using the NcoI and
XhoI restrictions sites, as described previously (17). The
pg-Vif expression vector was a generous gift of M. Malim (20).
SH3 and Kinase Vectors--
Hck cDNA was a kind gift
of K. Saksela (Finland) and was subcloned as an
EcoRI-XhoI fragment in the pCDNA3 vector
(Invitrogen). The plasmidial constructs pGST-SH3(Itk), pGST-SH3(Tec),
pGST-SH3(Lck), pGST-SH3(Hck), pGST-SH3(Crk), and pGSTSH3(Grb2) were
previously described (21). pGST-SH3(Yes) was generated as follows;
the SH3 domain of Yes was first amplified from H9 cell RNA extracts by
RT-PCR with the primers Yes-GST (s) and Yes-GST (as), and amplified DNA
was further cloned into pGEX-5X2 expression vector (Amersham Pharmacia
Biotech) at the EcoRI and NotI sites. The
pGST-SH3(Hck) W93A was obtained by the two-step recombinant PCR
methodology with the following primers, Hck-SH3 W93A (s/as),
GST-SH3(Hck) BamHI (s), and GST-SH3(Hck) NotI
(as). The pGST-SH3(Hck) W93F was a generous gift from M. Matsuda (22).
The DNA fragments of the SH3 domain mutants were amplified by PCR using
the primers GST-SH3(Hck) BamHI (s), GST-SH3(Hck)
BamHI Y68A (s), GST-SH3(Hck) BamHI D75A (s), and
GST-SH3(Hck) NotI (as) and cloned
BamHI/NotI into the pGEX-5X2 expression vectors.
Oligonucleotides
The oligonucleotides used for protein in vitro
translation were as follows: T3 Sam68 (s),
5'-ATTATGCTGAGTGATATCCCGCTATGAGCCGGTCCTCGGGCCGAG-3'; Sam-stop (as),
5'-ATAAGAATCTTGAACCTCCCCATG-3'; T3 Vif 1 (s),
5'-GTTATTAACCCTCACTAAAGGGAAGATGGAAAACAGATGGCAGGTGATG-3'; T3 Vif 64 (s),
5'-TATTAACCCTCACTAAAGGGAAGATGGTAGTAACAACATATTGG-3'; T3 VIF 128 (s),
5'-TATTAACCCTCACTAAAGGGAAGATGGTAGTCCTAGTTGTGAG-3'; Vif-stop 64 (as),
5'-TTACAGTCTAGTTTCTCCTAGTGG-3'; Vif-stop 128 (as),
5'-TTATATATGTCCTAATATGGCTTTTC-3'; Vif-stop 192 (as),
5'-ATTCTGCTATGTTGACAC-3'.
The cloning oligonucleotides used were synthesized as
following: GST-SH3(Yes) NotI (s),
5'-AAAAGAATTCCCGTTACTATATTTGTGGCCTTATATGATTATG-3'; GST-SH3(Yes)
EcoRI (as), 5'-AAAAAGCGGCCGCTAGTTTCAGCATCTTTTCTCCCC-3'; Vif-PPLA (s), 5'-CCACCTTTGGCCAGTGTTAGGAAG-3'; Vif-PPLA (as),
5'-CTTCCTAACACTGGAC AAAGGTGG-3'; Vif-AALA (s),
5'-GCTGCTTTGGCTAGTGTTAGGAAGCTAACAGAA G-3'; VIF-AALA (as),
5'-AGCCAAAGCAGCCTTTATCTTTTTTGGTG-3'; GST-SH3(Hck) BamHI (s),
5'-CGGGATCCCCGGCTCTGAGGACATCATCGTGGTT-3'; GST-SH3(Hck) NotI
(as), 5'-ATAGTTTAGCGGCCGCTAGCCCTTGAAAAACCACTTCTTTGTCTCC-3'; GST-SH3(Hck) BamHI Y68A (s),
5'-CGGGATCCCCGGCTCTGAGGACATCATCGTGGTTGCCCTGTATGATGCCGAGGCC-3'; GST-SH3(Hck) BamHI D75A (s),
5'-CGGGATCCCCGGCTCTGAGGACATCATCGTGGTTGCCCTGTATGATTACGAGGCCATTCACCACGAAGCCCTCAGC-3'; Hck-SH3 W93A (s), 5'-GAATCCGGGGAGGCGTGGAAGGCTCG-3'; Hck-SH3 W93A (as), 5'-CGAGCCTTCCACGCCTCCCCGGATTCC; GST-Vif NcoI (s),
5'-AATTCGCCATGGCTTCCCCTATACTAGGTTATTGGAAAA-3'; GST-Vif XhoI
(as), 5'-ATACGACTCGAGCTAATGTCCATTCATTGTATGGC-3'.
Transfections
Transfections of adherent cells were performed by the Fugen
method (Roche Molecular Biochemicals), and suspension cells were electroporated using the Gene Pulser II (Bio-Rad) as recommended by the
manufacturer. Briefly, for adherent cells, 293 cells were plated at
3.5 × 106 cells/75-cm2 flask and grown
overnight. Cells were incubated with 32 µl of Fugen and a total of 15 µg of appropriate plasmid DNAs for 72 h. Virus was quantified in
cell-free supernatants by measuring reverse transcriptase (RT) activity
(23) or by p24 enzyme-linked immunosorbent assay as recommended by the
manufacturer (Coulter). For stable transfection of Hck, 107
Jurkat cells were electroporated (250 mV, 960 microfarads) with 20 µg
of the pCDNA3-Hck construct. 24 h after transfection, G418 (2 mg/ml) was added to the cell culture followed by limiting dilution in
flat-bottomed 96-well plates. Hck expression was controlled in the
selected clones by Western blotting.
Expression and Purification of GST Fusion Proteins
Escherichia coli Top10 cells (Invitrogen) or BL21
codon plus RIL (Stratagene) transformed with fusion protein expression
plasmids were grown at 37 °C except for GST-Vif and GST-Gag, which
were grown at 30 °C. Protein expression was induced at an optical
density at 590 nm of 0.5-0.7, with 0.1-1 mM
isopropyl- In Vitro Transcription and Translation
For in vitro transcription, appropriate genes were
amplified by PCR using 5'-oligonucleotides that contain the T3 RNA
polymerase promoter upstream of the initiation position and
3'-oligonucleotides that contain a stop codon. PPLA and AALA mutant
vif genes were obtained by a two-step PCR on pNDK using,
respectively, Vif-PPLA and Vif-AALA (s) and (as) primers. Amplified
DNAs were subjected to in vitro transcription-translation
using the TNT coupled wheat germ extract system (Promega) as
recommended by the manufacturer. Proteins were translated in the
presence of [35S]methionine (1,000 Ci/mmol; Amersham
Pharmacia Biotech), resolved on 12% SDS-polyacrylamide gels, and
quantified by autoradiography and phosphorimager analysis.
Preparation of Cell Lysates for GST Pull-down
U937 promonocytic cells were washed twice in PBS and lysed in
Hepes buffer (1 ml/108 cells) containing 10 mM
Hepes, pH 7.0, supplemented with the protease inhibitor mixture
described above. Nucleic acids and insoluble materials were removed by
centrifugation at 15,000 × g for 15 min at
4 °C.
In Vitro Protein-Protein Interactions
Binding reactions were performed overnight at 4 °C in TBST
binding buffer containing 50 mM Tris-HCl, pH 7.0, 0.2%
Tween 20, and appropriate concentrations of NaCl (150-350
mM) in the presence of bovine serum albumin (200 µg/ml)
in a total volume of 300 ml. For the interaction with cytoplasmic Hck,
the incubation buffer was the same as used for preparation of cell
lysate. Agarose beads coupled to the GST fusion protein were incubated
overnight in 300 µl with either 8 µl of in vitro
translated 35S-labeled proteins or 200 µl of cytoplasmic
extract and then extensively washed in TBST buffer. Samples were
resuspended in 25 µl of SDS-PAGE sample buffer containing 5%
Vaccinia Virus Expression
Human U937 cells (1 × 107) were infected for
1 h with 1 plaque-forming unit of recombinant vaccinia virus/cell
to express T7 polymerase and then transfected with 20 µg of Pos7,
pCDNA Hck, or Pos7-Vif, using the electroporation technique, as
recommended by the manufacturer (Bio-Rad). Cells were harvested 24 h after transfection.
Antibodies
Mouse monoclonal antibodies used were anti-phosphotyrosine 4G10
(Upstate Biotechnology), anti-Lck 3A5 (Santa Cruz Biotechnology), anti-Hck H28520 (Transduction Laboratory), and anti-Fyn sc-434 (Santa
Cruz Biotechnology). Rabbit polyclonal antibodies used were anti-Vif
(gift of D. Gabuzda; Ref. 24), anti-Hck N30 (Santa Cruz Biotechnology),
and anti-GST sc-459 (Santa Cruz Biotechnology). The human anti-HIV
serum was a gift from J. Coniaux (Pasteur Institute, Belgium).
Western Blot Analyses
Following SDS-PAGE, proteins were electrotransferred to
polyvinylidene difluoride membranes (PerkinElmer Life Sciences).
Blots were saturated with 1% bovine serum albumin in TBST or in PBS (for 4G10 antibody) and incubated with appropriate primary antibodies (rabbit anti-Vif (1:3,000), mouse anti-Hck (1:1,000), mouse
anti-phosphotyrosine 4G10 (1:1000), mouse anti-Lck (1:1000), or rabbit
anti-GST (1:2000) antibodies). The secondary immunoreactions were
performed by using horseradish peroxidase-linked anti-rabbit,
anti-human, or anti-mouse immunoglobulins (Dako, 1:5,000), and followed
by ECL detection (Amersham Pharmacia Biotech).
Infection and Virus Propagation
Recombinant viruses were harvested 48 h after transfection
of HeLa cells with appropriate pNDK WT or vif In Vitro Kinase Assay
Human U937 (1 × 108) cells were lysed in 25 mM Hepes, pH 7.0, containing 1% Nonidet P-40, 100 mM NaCl, and 1 mM orthovanadate before
clarification. Hck was then immunoprecipitated by using rabbit anti-Hck
antibodies and protein G-Sepharose. After several washes in lysis
buffer containing 0.1% SDS, similar amounts of immunoprecipitated Hck
were incubated in kinase buffer (25 mM Hepes, pH 7.0, 100 mM NaCl, 10 mM MgCl2, 5 mM MnCl2) in presence of 5 µg of denatured
enolase (Sigma), [ Vif of HIV-1 Interacts Specifically with the SH3 Domain of
Hck--
SH3 domains recognize especially proline-rich motifs. Since
the Vif protein of HIV-1 contains a conserved PPLP motif in its C-terminal domain (Fig. 1), we
addressed the question whether Vif could bind SH3 domains from
different proteins. Eight different SH3 domains of tyrosine kinases and
adaptor proteins were expressed as fusion GST proteins in bacteria.
Following purification, by affinity chromatography on GSH-agarose
beads, GST fusion proteins were incubated with in vitro
translated [35S]Vif. After pull-down, bound
35S-labeled Vif protein was resolved by SDS-PAGE and
revealed by autoradiography. As shown in Fig.
2A, 35S-labeled
Vif protein binds to GST-SH3(Hck) but not to the control GST. In
contrast, the GST-linked SH3 domains of two members of the Src tyrosine
kinase family (Lck and Yes), or the Tec kinase family members (Itk and
Tec) or two adaptor proteins (Grb2, CrkII) failed to pull-down Vif. To
assess the functional activity of all GST-SH3 constructs, we measured
the binding of Sam68, a proline-rich protein, previously reported to
interact with most SH3 domains of different tyrosine kinases and
adaptor proteins. Sam68 bound strongly to the SH3 domains of Hck and
Lck tyrosine kinases and Crk II and Grb2 adaptor proteins (Fig.
2A). A weaker interaction between Sam68 and the SH3 domains
of Itk and Tec SH3 was also observed. Finally, Sam68 interacted very
faintly with the Yes SH3. Coomassie Blue staining of individual GST
chimera showed that the binding capacities of Vif and Sam68 were not
dependent of different stabilities and quantities of individual GST
fusions (Fig. 2A). The strength of the interaction between
Vif and Hck SH3 domain was further assessed using increasing NaCl
concentrations during the binding and washing steps. Interestingly, Vif
binding to the GST-SH3 of Hck was enhanced at 350 mM NaCl
compared with 150 mM NaCl concentration (Fig.
2B), suggesting that hydrophobic residues participated to
the interaction. These data indicate that Vif is recognized
preferentially by the SH3 domain of the tyrosine kinase Hck.
To exclude a possibility that the recognition of Vif by GST-SH3(Hck)
was due to a particular conformation and accessibility of the SH3
domain in the context of the GST fusion protein, we performed the
reverse experiment in which Vif was expressed as a GST-fusion protein.
U937 promonocyte cell lysate, endogenously expressing Hck, was
incubated with GST-Vif immobilized on GSH-agarose beads for pull down
assay. The presence of Hck was analyzed by Western blotting with
anti-Hck antibody. As shown in Fig.
3A, full-length Hck retains
its ability to bind GST-Vif but not GST, confirming the specificity of
the interaction.
The Vif/Hck interaction was then analyzed when both Hck and GST-Vif
have been expressed in human cells. GST-Vif and Hck were subcloned in
plasmids under the control of T7 polymerase promoter. GST-Vif and
Hck were then co-expressed in human promonocytic cells previously
infected with recombinant vaccinia virus expressing T7 polymerase.
Twenty hours after transfection, cell lysates containing GST fusion
proteins were precipitated by addition of GSH-agarose beads and bound
Hck protein was detected by Western blotting with anti-Hck antibody
(Fig. 3B). Co-precipitation of Hck with GST-Vif but not with
the control GST protein confirmed that Vif and Hck interact in a
specific manner in human cells.
Mapping of the Hck/Vif Interaction Domains--
The binding
affinity of proline motifs, including those present in HIV-1 Nef
protein, depends on hydrophobic interactions between residues of the
SH3 domain (such as Tyr-66, Tyr-68, and Trp-93 in Hck), but also on
ionic interactions between a basic residue found before or after the
proline motif and a highly conserved acidic residue in the SH3 domain
(Asp-75, in Hck) (25-27). Mutation of various residues of Hck SH3,
known to be important for the interaction between SH3 domains and their
specific ligands, were generated within GST fusion proteins (Y66A,
Y68A, and W93F or W93A, and D75A) and analyzed by pull-down assays
(Fig. 4). Most single mutations in the
SH3 domain of Hck, except the most conservative one (W93F), resulted in
the decrease of the interaction between Vif and Hck SH3 domain.
Coomassie Blue staining of Hck GST-SH3 mutants indicated that the
binding capacity of Vif was not dependent of different stabilities and
quantities of individual GST fusions (Fig. 4). Altogether, these
observations confirm that both hydrophobic and ionic residues of the
SH3 domain participate in the interaction with Vif protein.
We then mapped the domain of Vif interacting with the SH3 domain of
Hck. For this purpose we analyzed the binding of deleted mutants of Vif
protein with the SH3 domain of Hck. Vif protein was truncated in three
domains and expressed by in vitro translation. After
incubation with the GST or GST-SH3(Hck) constructs, bound proteins were
separated on SDS-PAGE and analyzed by phosphorimager. Fig.
5 reveals that the three thirds of Vif
may contribute to the interaction with the Hck SH3 domain, with a
weaker participation of the N-terminal third. We tested whether the
PPLP conserved proline-rich motif located in the C-terminal third of
Vif participates in the interaction with Hck SH3 domain. We produced
in vitro 35S-labeled Vif proteins in which the
PPLP motif was substituted by PPLA or AALA (Fig. 5). Pull-down
analysis, using GST-SH3(Hck), reveals that mutations of proline in the
C-terminal third domain of Vif decreased significantly the binding of
Vif to the SH3 domain of Hck. In contrast when PPLP was substituted to
AALA in the context of full-length Vif protein, the SH3 domain of Hck
was still able to interact. This observation confirms that other
domains of Vif participate in the interaction with the Hck SH3 module.
Moreover, we were unable to identify any point mutants of the
full-length Vif strongly affected in their binding to the SH3 domain of
Hck. Altogether, our data indicate several domains distributed all along the Vif protein participate in the interaction of Vif with the
SH3 domain of Hck, including the PPLP motif contained in the C-terminal
third of Vif.
Vif Inhibits Hck Activity and Is Not Itself a Substrate for
Hck--
To determine whether the interaction between Vif and Hck
contributes to the control of the Hck kinase activity, we performed a
kinase assay. For this purpose, Hck and Vif were transiently co-expressed in human promonocytic cells. Cell lysate analysis with
anti-Hck antibody revealed that the expression level of Hck in the
presence or in the absence of Vif was similar (Fig.
6A). To detect the
phosphorylation of Hck on tyrosine residues, we reprobed the Western
blot with anti-Tyr(P) antibody. When Hck was expressed alone, a band
corresponding to the phosphorylated Hck was detected, whereas Hck was
only weakly phosphorylated in presence of HIV-1 Vif. Moreover, we did
not detect phosphorylated proteins migrating at the expected position
of Vif suggesting that Vif is not phosphorylated on tyrosine by Hck. To
analyze whether Vif could have a direct effect on the Hck kinase
activity, we performed an in vitro kinase assay.
Cell-derived Hck immunoprecipitated from U937 cell lysate was incubated
with increasing concentrations of GST-Vif or GST. The catalytic
activity of Hck was followed both by autophosphorylation of Hck and by
transphosphorylation of exogenously added enolase (Fig. 6B).
The kinase activity of Hck was unchanged upon incubation with
increasing amounts of GST. By contrast, GST-Vif down-regulated both Hck
auto- and transphosphorylation activities in a
dose-dependent manner. In these experiments, no signal
corresponding to the phosphorylation of GST-Vif could be detected,
consistent with the idea that Vif is not a substrate for Hck. Together,
these observations suggest that Vif represses the tyrosine
phosphorylation activity of Hck in human cells.
Cells That Express Hck Display Attenuated Replication of
Vif Inhibition of HIV-1 Replication by Expression of Hck Is Overcome by
Vif--
To address the possibility that the Src tyrosine kinase Hck
is involved in the regulation of HIV-1 replication in a
Vif-dependent manner, several clones of Jurkat T cells
expressing constitutively Hck were established by stable transfection.
WT HIV-1 and vif
We next investigated virus production and infectivity during one single
virus cycle in cells expressing or not Hck. Human 293 cells were
co-transfected with plasmids expressing WT and vif
In the present work, we have investigated the putative interaction
between various SH3 domains and HIV-1 Vif protein. We identified a
preferential recognition of Vif by the SH3 domain of Hck and by the
full-length Hck tyrosine kinase. The functional consequence of this
interaction is the regulation of the HIV-1 replication in a
Vif-dependent manner in Hck-expressing cells. Indeed, we demonstrated that HIV-1 vif Our data suggest that Vif and Hck interact directly through the SH3
domain of Hck. Mutagenesis analysis revealed that conserved hydrophobic
(Tyr-66, Tyr-68, Trp-93) and charged residues (Asp-75) of the SH3
domain participate in the interaction with Vif protein. Those residues
were previously shown to play a crucial role for the interaction
between SH3 domains and their specific ligands (25-27). Since
proline-rich motifs are well known to interact with the SH3 domains, we
next attempted to identify the role played by the PPLP motif in the
recognition of the SH3 domain of Hck. When PPLP was mutated into PPLA
or AALA, the interaction of the C-terminal third of Vif with Hck SH3
was decreased. In contrast, when PPLP was substituted into AALA within
the full-length Vif protein, the interaction of Vif with the SH3 domain
of Hck was not abrogated. We concluded that the PPLP motif may be
necessary but not sufficient for interacting with the SH3 domain of
Hck. This is consistent with our observation showing that the central third of Vif binds to Hck SH3; the N-terminal third of Vif binds also
with Hck SH3 but at a lower extent. Interestingly, this proline-rich 151-164 region of Vif was also shown to be important for Vif
multimerization (28), and the replication of AALA Vif mutated HIV-1 was
completely abolished in restrictive
cells.2 Altogether, these
observations indicate that this region of Vif is involved in several functions.
Previous studies revealed also that Vif binds to the NCp7 domain of the
Gag protein of HIV-1 (17, 29, 30), and colocalizes with Gag in
virus-producing cells (16). However, Vif is not incorporated into viral
particles (18). Since the Src tyrosine kinases are located at the inner
layer of the plasma membrane in detergent-insoluble glycolipid-enriched
microdomains (31-33) and because HIV-1 buds preferentially from these
membrane regions (34), it will be interesting to determine whether Hck
controls the retention of Vif within cells during the virus budding.
We have observed that Vif inhibits the kinase activity of Hck both
in vitro and in promonocytic U937 cells. Moreover, the expression of Hck in absence of Vif induces a decrease of the viral
particle release as well as a significant loss of infectivity. Since
Vif has been reported to control functionally the processing of Gag in
restrictive T cells and in monocytic cells (6, 14, 35), it is tempting
to speculate that Vif controls the tyrosine phosphorylation of the Gag
precursor and therefore may also indirectly control the processing of
Gag. Further studies will be necessary to determine whether Gag is
phosphorylated on tyrosine by Hck in infected cells and to analyze the
consequences of these post-translational modifications on the viral
production and on the infectivity.
Hck is expressed mainly in promonocytic cells, including
monocyte-derived macrophages, in which it mediates relevant functions such as Fc In conclusion, Hck corresponds to an inhibitor of HIV-1 replication in
monocytic cells. It is the first cellular protein identified as
interacting with the auxiliary HIV-1 protein Vif. This interaction between Vif and Hck relieves the inhibition of HIV-1 replication and is
reminiscent of the antiviral activity described in T cells by others
(11, 12). The fact that Hck belongs to the Src family of tyrosine
kinases opens new areas of investigation on the role of tyrosine
kinases in the replication of HIV-1 and on the development of drugs
against HIV-1, possibly through the Vif/Hck binding interface.
replication was delayed in Jurkat T cell clones stably
expressing Hck. Our data demonstrate that Hck controls negatively HIV-1
replication and that this inhibition is suppressed by the expression of
Vif. Hck, which is present in monocyte-macrophage cells, represents the
first identified cellular inhibitor of HIV-1 replication
overcome by Vif.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) mutants are several orders of magnitude less
infectious than wild-type (WT) HIV-1 virions (1, 3-5). This critical
role of Vif was also observed in primary macrophages, where both
production of viral particles and infectivity of vif
viruses are reduced (6). The importance of Vif has
been confirmed in animal studies where vif-deleted simian
immunodeficiency viruses were drastically impaired in their propagation
and highly attenuated in their pathological properties (7, 8).
viruses in a single replication cycle, whose
properties are indistinguishable from those of WT viruses. Intermediate
phenotypes were also reported in U937 cells and primary macrophages.
Those semipermissive cells support the replication of vif
HIV-1 at reduced levels. Using a fusion technique
between permissive and restrictive cells, Simon et al. (11) and Madani
and Kabat (12) demonstrated that the restrictive phenotype is dominant over the permissive phenotype. These findings suggest that restrictive cells contain a potent activity inhibiting HIV-1 replication, which can
be counteracted by the expression of Vif.
HIV-1 replication.
In the absence of Vif, constitutive expression of Hck rendered T cells
less permissive for vif
HIV-1 replication.
Moreover, in cells expressing Hck, release of HIV-1 viral particles was
strongly inhibited and virion infectivity was reduced, when Vif was
lacking. Therefore, Hck represents the first identified cellular factor
that inhibits the replication of HIV-1 and that is overcome by Vif.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside for 3 h at
30 °C. The bacteria were then centrifuged at 5,000 × g for 15 min, and the pellet was frozen at
80 °C,
thawed in phosphate-buffered saline (PBS), and gently mixed for 10 min
at 4 °C. Bacteria were then lysed by sonication (3 × 30 s) on ice, and the lysate was incubated for 30 min at 4 °C in the
presence of 1% Triton X-100 with shaking. Insoluble material was
pelleted for 30 min at 14,000 × g, and the supernatant
was incubated overnight at 4 °C with 20 µl of 50% (v/v)
glutathione (GSH)-agarose beads (Sigma) added per ml of lysate. After
three washes in 1 M NaCl and in PBS, the GST fusion
proteins immobilized on GSH-agarose beads were quantified by
electrophoresing an aliquot on a 12% or 15% sodium dodecyl sulfate
(SDS)-polyacrylamide gel. The beads were stored no more than 1 week at
4 °C in the presence of protease inhibitor mixture (1 mg/ml
aprotinin, 1 mg/ml leupeptin, 2 mg/ml pepstatin, and 1 mg/ml antipain),
for further analysis.
-mercaptoethanol. Bound proteins were analyzed by SDS-PAGE, followed
by autoradiography or by Western blotting.
molecular clones. Virus stocks were then amplified by acute infection of SupT1 cells. Virus present in cell-free supernatants was quantified in C8166 indicator cells as described previously (15). Cells were
infected by incubating 2 × 106 cells in 1 ml of virus
supernatant at 37 °C with gentle shaking. After centrifugation at
800 × g for 5 min, cells were resuspended at 5 × 105/ml in culture medium. Virus replication was assayed
twice a week by determining the RT activity in the cell-free
supernatant as described (23). Virus infectivity was titrated on the
HeLa-derived P4 indicator cell line as described previously (15).
-32P]ATP (150 Ci/mmol), and
increasing concentrations of either GST or GST-Vif. After 30 min of
kinase reaction, the samples were separated by SDS-PAGE and analyzed by
autoradiography. The autophosphorylation activity of Hck and the
enolase phosphorylation were then quantified by phosphorimager analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Consensus sequence alignment of the Vif
proteins from different subtypes of HIV-1. The consensus sequences
from different HIV-1 M and O subtypes, coordinates 89-192 (C-terminal
half), were aligned using ClustalW software. The consensus amino acid
residues are conserved in at least 60% of the Vif sequences. The
symbol X indicates amino acid residues that are
not consensus (less than 60%). The Vif sequences were extracted from
1998/1999 HIV and SIV alignments at Los Alamos HIV sequence data base.
B1 and B2 sequences represent resulting consensus
on 86 and 88 sequences of B subtype, respectively. Both cysteines 114 and 133 and 161PPLP164 are indicated by their
coordinates.
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Fig. 2.
Vif interacts specifically with Hck SH3
domain. GST-SH3 pull-down of Vif protein. Five µg of the
appropriate GST-SH3 fusion protein or GST alone were incubated with
35S-radiolabeled Vif or Sam68, washed in buffer containing
150 mM NaCl, eluted, and separated by SDS-PAGE. The GST
fusion proteins were stained by Coomassie Blue, and pulled-down
proteins were revealed by autoradiography. Panel
A shows one representative interaction experiment. Bound
radiolabeled Vif protein was subsequently quantified by using a
phosphorimager. The bar graph at the
bottom (panel B) presents the mean
percentages of Vif binding to GST fusion proteins at 150 and 350 mM NaCl, compiled from six independent pull-down
experiments.
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Fig. 3.
GST-Vif interacts specifically with Hck.
A, Hck expressed in U937 promonocytes interacts with
GST-Vif. U937 (107 cells) were lysed in 1% Nonidet P-40,
10 mM Hepes, pH 7.0, and clarified before pull-down assay
with 10 µg of GST or GST-Vif. Hck interacting with GST-Vif was
separated by SDS-PAGE and revealed by Western blot analysis.
B, GST-Vif and Hck coprecipitate in U937 cells. Human U937
cells (107 cells) were infected with vaccinia virus
expressing the T7 polymerase before electroporation with pos7-GST or
pos7-GST-Vif and pCDNA-Hck. Twelve hours after infection, the
infected cells were lysed in 1% Nonidet P-40, 10 mM Hepes,
pH 7.0. Following clarification, GST and GST-Vif present in the
supernatant were precipitated with glutathione-agarose beads. The
precipitation products were separated by SDS-PAGE and analyzed by
Western blotting against GST or Hck. We simultaneously assayed the Hck
expression level by direct Western blot analysis of the cell
lysate.
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Fig. 4.
Mutations in the Hck SH3 domain affecting its
interaction with the Vif protein. Five point mutants of the Hck
SH3 domain fused to GST were generated. The corresponding open reading
frames are shown in the upper amino acid alignments. Five µg of
GST-SH3 fusion protein or GST alone were incubated with
35S-radiolabeled Vif protein, for pull-down assays (150 mM NaCl). Radiolabeled-Vif protein bound to the SH3 domains
was separated by SDS-PAGE. The GST fusion proteins were stained by
Coomassie Blue, and pulled-down proteins were revealed by
autoradiography.
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Fig. 5.
Domains of Vif interacting with Hck SH3
domain. Full-length vif gene or its fragments were
amplified by PCR and were translated in vitro, using wheat
germ extracts, in the presence of [35S]methionine. The
binding of Vif proteins to GST or GST-Hck(SH3) was revealed by
autoradiography.
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Fig. 6.
Vif controls the Hck tyrosine kinase
activity. A, Vif inhibits Hck tyrosine phosphorylation
in U937 promonocytes. Human U937 cells infected by vaccinia virus
expressing the T7 polymerase were transfected with pos7-Vif or
pos7-Hck. Twelve hours after infection, the infected cells were lysed
in 1% Nonidet P-40, 10 mM Hepes, pH 7.0, and cell lysate
was analyzed by SDS-PAGE. Appropriate proteins were revealed by Western
blotting with antibodies against Tyr(P), Hck, or Vif. B,
GST-Vif down-regulates kinase activity. Hck was immunoprecipitated from
(1 × 108) U937 cells. After washes, equal amounts of
Hck were incubated in kinase buffer in the presence of 5 µg of
denatured enolase, [ -32P]ATP, and increasing
concentrations of either GST or GST-Vif (from 1 to 10 µg). After 30 min of kinase reaction, the samples were separated by SDS-PAGE and
analyzed by autoradiography. The autophosphorylation activity of Hck
was then quantified by phosphorimager.
HIV-1--
Human cells were previously classified as
restrictive (H9, T CD4+ cells), semipermissive (U937 cells,
macrophages), or permissive (CEM, SupT1, Jurkat, C8166) for vif
HIV-1. We investigated whether a correlation may
exist between the presence of Hck and the level of replication of
HIV-1. In absence of Vif, as shown in Fig.
7, comparative analysis of the expression
of Hck within human cells known to be infected by HIV-1 revealed that
permissive cell lines lack the expression of both isoforms of Hck,
p59hck and p61hck. In
contrast, restrictive and semipermissive cells expressed Hck, except
for the restrictive primary T CD4+ lymphocytes which were negative for
Hck. Since all Hck-positive cells displayed an attenuated phenotype of
vif
viruses, this suggests that Hck
expression may participate to the control of HIV-1 replication in a
Vif-dependent manner.
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Fig. 7.
Comparative analysis of the expression level
of Hck within human T cells and monocytes/macrophages. Two million
T cells, promonocyte cells, and primary T cells and adherent
macrophages were cultured before lysis and clarification. After SDS gel
electrophoresis, the expression level of Hck was analyzed by Western
blot using an anti-Hck monoclonal antibody. The bottom of
the panel indicates the level of Vif- HIV-1 replication
within the different cell types analyzed. +, permissive cells where
replication was complete; D, semipermissive cells where
replication was delayed; , restrictive cells where no replication
occurred.
replications were compared
in both cell lines by measurement of the virus-associated RT activity
in the supernatant of infected cells (Fig.
8A). The replication of
vif
HIV-1 was delayed in Jurkat cells stably
expressing Hck, as compared with parental Jurkat cells. WT virus
replicates efficiently in both Jurkat and Hck expressing Jurkat cells.
A much more significant delay of vif
HIV-1
replication was observed in U937 promonocytes, endogenously expressing
Hck (Fig. 8B). Interestingly, in both Jurkat Hck and in U937
cells, persistent infections were established with WT and vif
virus. Altogether, these viral replication data
suggest that Hck delays vif
virus
replication.
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Fig. 8.
A, Hck tyrosine kinase expression
down-regulates the HIV-1 vif-deficient virus replication in
Jurkat cells. Stably transfected Jurkat T cells expressing Hck
(Jurkat Hck) and untransfected Jurkat cells were
assayed for Hck expression and for Lck expression by Western blotting
shown in the right part of the figure. Jurkat and Jurkat Hck
cells were infected with WT ( ) or vif
(
) HIV-1 at a multiplicity of infection of 0.05 (as measured by
titration in C8166 cells). The replication kinetics was monitored by
measuring RT activity in the supernatant of infected cells twice weekly
for 21 days. Data represent the infections of one Jurkat Hck clone are
means with standard errors of RT activities among triplicate parallel
infections. These results were reproduced with two other Jurkat Hck
clones. B, delay of vif
deficient
virus replication in U937 promonocytic cells. U937 cells were infected
with WT (
) or vif
(
) HIV-1 at a
multiplicity of infection of 0.05 titrated in C8166 cells and the
replication was monitored as described in panel A
for 50 days. The curve shown corresponds to one representative
experiment.
virus together with WT Hck or Hck carrying a mutation in
its SH3 domain (W93A Hck) that dramatically reduced the interaction between SH3 domain of Hck and Vif (Fig. 4). Both WT and mutated Hck
reduced the vif
virus production, measured
either by RT activity (Fig.
9A) or by enzyme-linked
immunosorbent assay p24 (data not shown), down to 5% (WT) and 13%
(W93A Hck) of the virus production observed in Hck-negative cells. In
contrast, WT virus production was not significantly affected by the
expression of WT Hck, suggesting that Vif can overcome the negative
effect induced by Hck expression. Interestingly, W93A Hck mutant was
not counteracted by Vif expressed from WT HIV-1. Therefore, there is a
correlation between the alteration of Vif binding to the SH3 domain of
Hck and its inability to counteract Hck. Anti-Hck Western blot analysis
confirmed that the difference in virus production did not result from
variation of WT and W93A Hck expression level (Fig. 9B), and
in vitro kinase assays demonstrated that both WT Hck and
W93A Hck displayed similar kinase activities (Fig. 9C).
Furthermore, to confirm the role of Vif in the control of virus
production in presence of Hck, we analyzed the restoration of
vif
virus release by expressing in trans
HIV-1 Vif (pgVif). Human 293 cells were co-transfected with plasmids
expressing Hck and vif
HIV-1 in presence or
absence of pgVif. Virus production was increased up to 6-fold when Vif
was added in trans, confirming that Vif counteracts the
effect of Hck (data not shown). Since the vif
phenotype usually described in T lymphocytes is mainly a defect of
infectivity of neosynthesized virus, we next analyzed the infectivity of virus produced in the presence or in the absence of Hck.
Virus-containing supernatant from transfected cells was harvested and
titrated for infectivity by end point dilution assay. As shown in Fig. 9D, we observed that the infectivity of vif
virus was reproducibly 3-4-fold lower than that of WT
virus in Hck-expressing cells. We also compared the infectivity of
vif
and WT viral particles produced by
Jurkat-Hck cells and U937 cells, endogenously expressing Hck. The
viruses were collected at the peak of virus production and quantified
by RT activity measurement. Titration of virus infectivity, by end
point dilution assay, revealed that infectivity of vif
virus is decreased about 15-fold in Jurkat-Hck cells and
11-fold in U937 cells. All together, our results indicate that both the virus production and infectivity of vif
HIV-1
are highly decreased in Hck-expressing cells and that Vif is able to
counteract this inhibitory activity.
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Fig. 9.
Inhibition of
vif HIV-1 production and infectivity by
Hck. A, the production of viral particles is inhibited
by Hck. Human 293 cells were cotransfected with a plasmid containing
either WT (
) or vif
(
) HIV-1 provirus
together with a plasmid expressing Hck or Hck (W93A). The virus
production was monitored by RT activity measurement in the supernatants
of cell cultures. Data represent means of six independent experiments
with standard errors. B, Hck expression in 293 transfected cells. Human 293 cells, transfected as in panel
A, were lysed 48 h after transfection, and Hck
expression was analyzed by anti-Hck Western blotting. C, Hck
and W93A Hck were immunoprecipitated from transfected cells and
incubated in kinase buffer in the presence of
[
-32P]ATP for 30 min. After SDS-gel electrophoresis,
the autophosphorylation activity of Hck was analyzed by
autoradiography. D, Hck expression inhibits vif
HIV-1 infectivity. The HIV-1 stocks for the
infectivity assay were collected from transfected 293 cells as in
panel A. Following standardization of virus
stocks by RT activity, infectivity of WT and vif
viruses was scored on the HeLa-derived P4 indicator
cell line. Infectivity values correspond to the mean number of HeLa P4
blue cells counted for the same RT activities of WT and vif
virions. Data represent means of three independent
assays with triplicates.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
replication was
inhibited when Hck was stably expressed in permissive Jurkat T cells.
Similar effects on the replication kinetics were observed in U937
promonocytes (present work) and in primary macrophages infected by
vif
viruses (6). By analyzing HIV-1
production in one viral cycle, we have demonstrated that transient
expression of Hck decreased virus release and reduced the infectivity
of neo-synthesized viral particles. This last infectivity defect was
mostly documented in the context of HIV-1 vif
replication in primary T cells and in H9 cells. Vif counteracted this
inhibitory effect of Hck, when it was expressed in cis and trans. We further confirmed this inhibitory effect of Hck,
by analyzing the virus production of WT HIV-1 in cells expressing Hck
mutated in its SH3 domain, in order to relieve its interaction with Vif
of HIV-1. In these conditions, Vif lost its ability to counteract the
negative effect of Hck on HIV-1 virus production.
RI receptor signaling, induction of cytokine production triggered by bacterial lipopolysaccharide, phagocytosis, and cell spreading (36-38). Primary macrophages are only weakly permissive for
vif
HIV-1 and show a reduction of free virus
infectivity and, to a lesser extent, a decrease of viral production
(6). These two effects were reproduced in Hck-negative T CD4+ cells
transfected with a Hck-expressing plasmid. We observed also that Hck is
naturally present in H9 T CD4+ cells (Fig. 7), the only immortalized T
CD4+ cells which are restrictive for vif
HIV-1. Therefore, our data suggest that the presence of Hck may contribute to the reduced capacity of vif
HIV-1 to replicate in these cells. However, the undetectable level of
Hck in primary T CD4+ lymphocytes suggests that the vif
restriction phenotype could be dependent upon cellular
factors other than Hck in a cell type-dependent manner.
Since these cells express Src tyrosine kinases such as Lck, Fyn, Yes,
Src, and Fgr, with functions redundant with Hck, it will be important
to evaluate carefully the role of these kinases, alone or in
combination, in the inhibition of vif
HIV-1
in primary T cells. Moreover, the picture may be further complicated
since the expression level of Src kinases has been shown to be
modulated in function of the cellular activation state (39, 40).
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ACKNOWLEDGEMENTS |
---|
We thank I. Hirsch, J. Sire, G. Quérat, B. Canard, J.M. Ruyschaert, O. Schwartz, C. Mawas, J. Ewbank, and Q. Sattentau for critical review of the manuscript. We acknowledge M. Malim for the generous gift of pgvif and G. Sutter and B. Moss for the vaccinia virus/posT7 constructs.
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FOOTNOTES |
---|
* This work was supported in part by the following French institutes: INSERM (Dotation Globale INSERM), Agence Nationale de Recherche sur le SIDA (ANRS), and Ensemble contre le Sida (Sidaction).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.
§ Supported by Sidaction.
¶ These two authors contributed equally to this work.
** Supported by ANRS.
To whom correspondence may be addressed. Tel.:
33-4-91-82-75-83; Fax: 33-4-91-82-60-61; E-mail:
rvigne@inserm-u372.univ-mrs.fr.
¶¶ Supported by the Fondation Nationale de la Recherche Scientifique (Belgium). To whom correspondence may be addressed. Tel.: 33-4-91-82-75-83; Fax: 33-4-91-82-60-61; E-mail: edecroly@ulb.ac.be.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M009076200
2 G. Bessou, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: HIV, human immunodeficiency virus; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; WT, wild type; SH, Src homology; TBST, Tris-buffered saline with Tween 20; PCR, polymerase chain reaction; RT, reverse transcription; PBS, phosphate-buffered saline; s, sense, as, antisense.
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REFERENCES |
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