The Multimerization of Human Immunodeficiency Virus Type I Vif Protein

The Vif (virioninfectivity 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 ofvif-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. The Vif (virioninfectivity 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 ofvif-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. human immunodeficiency virus type I glutathioneS-transferase chloramphenicol acetyltransferase polymerase chain reaction polyacrylamide gel electrophoresis vesicular stomatitis virus The accessory genes of HIV-1,1 includingvif, vpr, nef, and vpu, have been shown to play important roles during HIV-1 infection (1Emerman M. Malim M.H. Science. 1998; 280: 1880-1884Crossref PubMed Scopus (314) Google Scholar). It has been demonstrated that Vif affects the late stages of the viral life cycle, possibly through the assembly of viral particles (2Gabuzda D.H. Lawrence K. Langhoff E. Terwilliger E. Dorfman T. Haseltine W.A. Sodroski J. J. Virol. 1992; 66: 6489-6495Crossref PubMed Google Scholar, 3Blanc D. Patience C. Schulz T.F. Weiss R. Spire B. Virology. 1993; 193: 186-192Crossref PubMed Scopus (52) Google Scholar, 4von Schwedler U. Song J. Aiken C. Trono D. J. Virol. 1993; 67: 4945-4955Crossref PubMed Google Scholar). The vif-defective (vif−) viruses are able to penetrate into target cells but not accomplish reverse transcription (4von Schwedler U. Song J. Aiken C. Trono D. J. Virol. 1993; 67: 4945-4955Crossref PubMed Google Scholar, 5Courcoul M. Patience C. Rey F. Blanc D. Harmache A. Sire J. Vigne R. Spire B. J. Virol. 1995; 69: 2068-2074Crossref PubMed Google Scholar, 6Sova P. Volsky D.J. J. Virol. 1993; 67: 6322-6326Crossref PubMed Google Scholar, 7Dornadula G. Yang S. Pomerantz R.J. Zhang H. J. Virol. 2000; 74: 2594-2602Crossref PubMed Scopus (39) Google Scholar). The requirement for Vif, however, is cell type-specific. Thevif− 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 (2Gabuzda D.H. Lawrence K. Langhoff E. Terwilliger E. Dorfman T. Haseltine W.A. Sodroski J. J. Virol. 1992; 66: 6489-6495Crossref PubMed Google Scholar, 4von Schwedler U. Song J. Aiken C. Trono D. J. Virol. 1993; 67: 4945-4955Crossref PubMed Google Scholar, 8Gabuzda D.H. Li H. Lawrence K. Vasir B.S. Crawford K. Langhoff E. J. Acquir. Immune Defic. Syndr. 1994; 7: 908-915PubMed Google Scholar). 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 (9Trono D. Cell. 1995; 82: 189-192Abstract Full Text PDF PubMed Scopus (242) Google Scholar). 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 (10D'Aloja P. Olivetta E. Bona R. Nappi F. Pedacchia D. Pugliese K. Ferrari G. Verani P. Federico M. J. Virol. 1998; 72: 4308-4319Crossref PubMed Google Scholar). 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 (11Simon J.H. Gaddis N.C. Fouchier R.A. Malim M.H. Nat. Med. 1998; 4: 1397-1400Crossref PubMed Scopus (246) Google Scholar, 12Madani N. Kabat D. J. Virol. 1998; 72: 10251-10255Crossref PubMed Google Scholar). 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 (13Simon J.H. Carpenter E.A. Fouchier R.A. Malim M.H. J. Virol. 1999; 73: 2667-2674Crossref PubMed Google Scholar). Although it has been demonstrated that Vif of HIV-1 interacts with the NCp7 domain of p55 Gag precursorin 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 (13Simon J.H. Carpenter E.A. Fouchier R.A. Malim M.H. J. Virol. 1999; 73: 2667-2674Crossref PubMed Google Scholar, 14Bouyac M. Courcoul M. Bertoia G. Baudat Y. Gabuzda D. Blanc D. Chazal N. Boulanger P. Sire J. Vigne R. Spire B. J. Virol. 1997; 71: 9358-9365Crossref PubMed Google Scholar, 15Simon J.H. Fouchier R.A. Southerling T.E. Guerra C.B. Grant C.K. Malim M.H. J. Virol. 1997; 71: 5259-5267Crossref PubMed Google Scholar, 16Huvent I. Hong S.S. Fournier C. Gay B. Tournier J. Carrière C. Courcoul M. Vigne R. Spire B. Boulanger P. J. General Virol. 1998; 79: 1069-1081Crossref PubMed Scopus (58) Google Scholar). 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 (31Zhang H. Pomerantz R.J Dornadula G. Sun Y. J. Virol. 2000; 74: 8252-8261Crossref PubMed Scopus (103) Google Scholar). 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. 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 (17Zhang H. Dornadula G. Alur P. Laughlin M.A. Pomerantz R.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12519-12524Crossref PubMed Scopus (43) Google Scholar). 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-VifΔ151–164 were constructed by replacing theHin dIII-Bam HI fragment (containing vp gene) of pSG5GalVP with a PCR-amplified complete vif gene or its mutant Δ151–164. The pVif-VP or pVifΔ151–164-VP were constructed by replacing the Eco RI-Bgl II fragment (containing gal4 gene) of pSG5GalVP with a PCR-amplified complete vif gene or its mutant Δ151–164, respectively (18Shimano R. Iida S. Fukumori T. Yamamoto Y. Kawamura M. Furuta R.A. Adachi A. Biochem. Biophys. Res. Comm. 1998; 242: 313-316Crossref PubMed Scopus (12) Google Scholar). The integrity of all the constructs was confirmed by DNA sequencing. 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 mmisopropylthio-β-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. Afterin 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. The35S-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. 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 (17Zhang H. Dornadula G. Alur P. Laughlin M.A. Pomerantz R.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12519-12524Crossref PubMed Scopus (43) Google Scholar, 19Zhang H. Duan L.X. Dornadula G. Pomerantz R.J. J. Virol. 1995; 69: 3929-3932Crossref PubMed Google Scholar). 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 mmphenylmethylsulfonyl 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 were to the respectively. The antibody or antibody (Research Antibodies, Santa Cruz, was as the A Pharmacia was to the For cell lysates from COS-1 or 293T cells expressing were incubated with anti-c-Myc antibody by at 4 °C, followed by with protein (Amersham Pharmacia for an 2 The was washed three with cell lysing buffer. The was then resuspended in SDS-containing buffer, at 95 °C, and centrifuged at 12,000 × The supernatant was then subjected to After onto a nylon/nitrocellulose the samples were with a mouse anti-FLAG (Research Antibodies, Santa Cruz, was as a A mammalian two system, which was from the two-hybrid was to study the self-association of HIV-1 Vif proteins in (18Shimano R. Iida S. Fukumori T. Yamamoto Y. Kawamura M. Furuta R.A. Adachi A. Biochem. Biophys. Res. Comm. 1998; 242: 313-316Crossref PubMed Scopus (12) Google Scholar, H. J. Virol. 1993; 67: PubMed Google Scholar). The was as with some (18Shimano R. Iida S. Fukumori T. Yamamoto Y. Kawamura M. Furuta R.A. Adachi A. Biochem. Biophys. Res. Comm. 1998; 242: 313-316Crossref PubMed Scopus (12) Google Scholar, H. J. Virol. 1993; 67: PubMed Google Scholar). 5 μg of pGal-Vif and pVif-VP were with into using the CA). 48 the cells were lysed in lysing buffer (Promega, Madison, WI) and subjected to a chloramphenicol acetyltransferase as H. Duan L.X. Dornadula G. Pomerantz R.J. J. Virol. 1995; 69: 3929-3932Crossref PubMed Google Scholar). The of Vif mutants was by using a viral infectivity with some G. Yang S. Pomerantz R.J. Zhang H. J. Virol. 2000; 74: 2594-2602Crossref PubMed Scopus (39) Google Scholar). HIV-1 H9 cells were transfected with 5 μg of (containing vesicular stomatitis virus and wild-type vif gene or its mutants by G. Yang S. Pomerantz R.J. Zhang H. J. Virol. 2000; 74: 2594-2602Crossref PubMed Scopus (39) Google Scholar, U. Trono D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar). The was by a gene and Hercules, CA). 10% was to the transfected H9 cells. the viral particles in supernatant were and pelleted via G. Yang S. Pomerantz R.J. Zhang H. J. Virol. 2000; 74: 2594-2602Crossref PubMed Scopus (39) Google Scholar). After by the HIV-1 which was via assays from the viruses were to 5 × cells M. Res. PubMed Scopus Google Scholar). 48 the cells were lysed in lysing buffer and subjected to Vif proteins have a tendency toward GST-Vif was in bacterial cells and onto glutathione-conjugated agarose In Vif proteins were to with the GST-Vif-conjugated The Vif was then analyzed by followed by direct autoradiography. 1 A that GST-Vif but not GST can to in Vif protein, indicating a Vif-Vif in HIV-1 Vif protein was directly loaded onto a gel (SDS-free) for with loading 10% only or SDS at various the native or relatively native conditions, the 35S-labeled Vif proteins in the Tris/glycine gels as and or 1 With the of of SDS in the loading buffer, the of Vif a the sample was at 95 °C for 5 min, all the multimers of Vif proteins suggesting that the Vif-Vif binding is to be It is that, to the sample in HIV-1 Vif protein was with RNase A to remove RNA the Vif-Vif binding should be the binding for Vif a of in Vif protein have been generated through followed by in vitro in the presence of These Vif mutants were then to to GST-Vif fusion protein to agarose 2 A that the deletion of the C terminus in Vif protein the Vif-Vif binding Further studies indicated that deletion at amino acid 151–164 this binding 2 A). This result was further confirmed by native In the presence of 0.1% Vif mutants and were unable to form multimers, other mutants were able to 2 It is that there are positively charged amino acids in the 151–164 The mutants that substitute positively charged amino generated by J. B. Yang Gabuzda D. J. Virol. 1995; 69: PubMed Google have been for this Vif-Vif However, all mutants contain Vif-Vif binding not It is also that there are and in this is in various of HIV-1 or immunodeficiency are important for Vif-Vif binding remains to be further the that Vif self-association also we a coimmunoprecipitation The Vif protein was tagged with c-Myc or FLAG at its C terminus, respectively, and in the COS-1 cells. that the expression of Vif and Vif could be via Western blotting, with mouse anti-c-Myc antibody or goat anti-FLAG respectively two study Vif-Vif the cell lysates were with antibody and then subjected to followed by Western The goat anti-FLAG antibody was to 3 demonstrated that the Vif was with Vif when mouse antibody was for the suggesting a Vif-Vif interaction within a cell the in vivo Vif-Vif interaction was by the mammalian two-hybrid A fusion protein of and is able to the function as a DNA binding function as a DNA HIV-1 Vif protein was to or respectively A). the interaction between Vif proteins the and domain be and the binding promoter be 4 indicated that, like Vif in fusion protein could to Vif in the fusion protein and the expression of As pGal-Vif or pVif-VP were unable to expression 3 and also that Vif mutant Δ151–164, which not have the to interact with Vif protein in other also could not interact with Vif in this As Vif in the late of HIV-1 life and is by nonpermissive cells, such as peripheral blood mononuclear cells, macrophages, and H9 (2Gabuzda D.H. Lawrence K. Langhoff E. Terwilliger E. Dorfman T. Haseltine W.A. Sodroski J. J. Virol. 1992; 66: 6489-6495Crossref PubMed Google Scholar, 3Blanc D. Patience C. Schulz T.F. Weiss R. Spire B. Virology. 1993; 193: 186-192Crossref PubMed Scopus (52) Google Scholar, 4von Schwedler U. Song J. Aiken C. Trono D. J. Virol. 1993; 67: 4945-4955Crossref PubMed Google Scholar). the of Vif we Vif mutant which is unable to form multimers in the and within cells, is able to Vif function in the viral life cycle. this a viral infectivity was Vif or its mutants were in the nonpermissive H9 T-cells. the HIV-1 without vif and in were generated from cells. After for the viruses were to the target cells which an expression the HIV-1 The viral infectivity was by the of gene expression in the target cells, which is by the HIV-1 protein by the synthesized 5 that, when the wild-type vif gene was in HIV-1 virus-producing nonpermissive H9 T-cells, the viral infectivity could a However, when was in HIV-1 virus-producing nonpermissive H9 T-cells, the viral infectivity was with the vif-defective HIV-1 viruses These indicated that the 151–164 deletion 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 that also demonstrated that this fragment is essential for Vif function J.H. Carpenter E.A. Fouchier R.A. Malim M.H. J. Virol. 1999; 73: PubMed Google Scholar). This demonstrated that multimerization of Vif proteins is for Vif HIV-1 including reverse and have been shown to form or multimers in vitro and in The of or multimers has been demonstrated to be important for in the life Biochem. 1998; 67: PubMed Scopus Google Scholar, F. Biochem. PubMed Scopus Google Scholar, S. J. 1994; Full Text PDF PubMed Google Scholar, N. S. K. C. S. R. J. Virol. 2000; 74: PubMed Scopus Google Scholar). In multimerization is to the of many and proteins and is a for the of In this study, we analyzed the multimerization of HIV-1 Vif proteins via various The in vitro-translated, Vif proteins were able to form multimers in the native Conversely, GST-Vif fusion GST which were generated from the bacterial expression system, were able to to the in vitro-translated, Vif coimmunoprecipitation and a mammalian two also demonstrated a Vif-Vif interaction These in vitro and in vivo the that Vif proteins are able to form As the deletion of the domain that is essential for the Vif-Vif binding 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 remains the role of Vif multimerization and the or 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 and proline-enriched fragment acid As the positively charged amino acids in this are not for the Vif-Vif the are important remains to be It is that a acid for is to this It has also been shown that is by the protein of Vif, and this is important for Vif function Gabuzda D. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). As are to the domain for it is that the multimerization of Vif proteins is by in the virus-producing cells. the positively charged amino acids in and in the C terminus of Vif are for binding in vitro M. Courcoul M. Bertoia G. Baudat Y. Gabuzda D. Blanc D. Chazal N. Boulanger P. Sire J. Vigne R. Spire B. J. Virol. 1997; 71: 9358-9365Crossref PubMed Google Scholar). Recently, we demonstrated that HIV-1 Vif is an RNA binding protein and an of a messenger ribonucleoprotein complex of viral RNA in the cytoplasm and could be involved in the viral RNA packaging (31Zhang H. Pomerantz R.J Dornadula G. Sun Y. J. Virol. 2000; 74: 8252-8261Crossref PubMed Scopus (103) Google Scholar). In to with NCp7 via its C terminus, Vif to RNA via its Although RNA to Vif to Gag at the when RNA is mixed with Vif or Gag RNA only to Gag but not Vif when Vif protein is mixed with RNA and NCp7 (31Zhang H. Pomerantz R.J Dornadula G. Sun Y. J. Virol. 2000; 74: 8252-8261Crossref PubMed Scopus (103) Google Scholar). This could be of various and is However, as the for Vif multimerization and for binding are in or possibly it is that the interaction between Vif and as as the Vif, and is by Vif the of Vif multimerization may be in understanding the of Vif protein, the molecular mechanism(s) of HIV-1 Vif in the viral life cycle. In a target for We J. A. and for of the and We also Gabuzda for plasmids of Vif mutants