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J. Biol. Chem., Vol. 280, Issue 19, 18573-18578, May 13, 2005
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From the Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaracho, Sakyo-ku, Kyoto 606-8507, the Department of Molecular Cell Biology, Graduate School of Medicine, Osaka City University, Osaka 545-8585, and CREST, Japan Science and Technology Corporation (JST), Kawaguchi, Saitama 332-0012, Japan
Received for publication, March 1, 2005 , and in revised form, March 18, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ubiquitin-mediated protein degradation system has been shown to be involved in a wide variety of cellular functions, including cell cycle progression and signal transduction (14). Ubiquitination is a multistep process of a three-enzyme cascade involving the ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin ligase (E3) enzymes (15). The E3 binds both the protein target and a cognate E2 to mediate the transfer of ubiquitin from the E2 to the substrate protein and plays a pivotal role in substrate recognition and conferring specificity to the ubiquitination pathway. Since a Vif-BC-Cul5 complex is an E3-like complex, it has suggested that Vif functions as a substrate recognition subunit of the Vif-BC-Cul5 complex and targets APOBEC3G for ubiquitination and subsequent degradation by the proteasome, resulting in inhibition of the incorporation of APOBEC3G into HIV-1 virions and protection of the viral DNA from mutation (10, 16, 17). However, there has been no direct evidence on the ubiquitination of APOBEC3G by a Vif-BC-Cul5 complex. Moreover, there still remain some controversies between the Vif-BC-Cul5 complex formation and/or substrate recognition by Vif and its activity to antagonize APOBEC3G. Here, we have first shown that a Vif-BC-Cul5 complex could indeed work as an E3 ligase complex by an in vitro ubiquitin conjugation assay using purified Vif-BC-Cul5 complex. By using the assay, we have shown that a Vif-BC-Cul5 complex induces the ubiquitination of the wild type (WT) APOBEC3G, but not that of D128K APOBEC3G, which does not interact with HIV-1 Vif (18, 19). Moreover, we have also shown the clear relationship between the ability of Vif to overcome APOBEC3G and the ubiquitination of APOBEG3G by the Vif-BC-Cul5 complex using several Vif mutants.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vif-Luc were constructed as described previously (8). Cell Lines—HEK293T cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% FCS and penicillin, streptomycin, and glutamine (PSG) (Invitrogen). M8166 cells were maintained in RPMI1640 (Sigma-Aldrich Corp.) containing 10% FCS and PSG.
Preparation of HIV-1 Virus and Infectivity Assay—Luciferase reporter viruses with or without Vif were prepared in HEK293T cells by cotransfection of pNL43-Luc (WT) or pNL43/vif-Luc (Vif) together with a mock vector or expression vectors for APOBEC3G WT or D128K and Vif WT or its mutants by calcium phosphate method as described previously (8). Viruses in the supernatants were collected after 48 h of transfection, and virus titers were measured with an enzyme-linked immunosorbent assay kit for the p24 antigen (RETRO-TEK, Zepto-Metrix Corp., Buffalo, NY). An adjusted amount of viruses was challenged to target cells, M8166. Twenty-two hours postinfection, the cells were lysed in passive lysis buffer (Promega, Madison, WI), and the luciferase activity was measured with a Luminometer (EG & G Berthold, Bad Wildbad, Germany). Values were presented as percent infectivity relative to the value of wild type virus without expression of APOBEC3G.
Co-immunoprecipitation Assay—To see protein-protein interaction in vivo, we performed an immunoprecipitation assay as described previously (8). Expression vectors for HA-APOBEC3G WT or D128K were cotransfected with expression vectors for Vif WT or its mutants into HEK293T cells by calcium phosphate method. Two days after transfection, cells were lysed in RIPA buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% deoxycholate). Complexes were immunoprecipitated with 1 µg of anti-HA monoclonal antibody (mAb) (12CA5) (F. Hoffmann-La Roche Ltd.) for 1 h, followed by addition of 20 µl of protein A-SepharoseTM beads (Amersham Biosciences) for 1 h at 4 °C. The beads were washed with RIPA buffer three times and analyzed on immunoblot with anti-HA mAb or rabbit antiserum against Vif (a kind gift from Dr. D. Gabuzda through the AIDS Research and Reference Reagent Program) (23). HA-APOBEC3G and Vif were visualized by ECL detection system (Amersham Biosciences).
Recombinant baculoviruses for Vif WT or its mutants were co-infected Hi Five cells with Cul5, EloB, EloC, and Rbx1 expressing recombinant viruses. Sixty-four hours after infection, cells were harvested and lysed in lysing buffer (0.5% Triton X-100, 25 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 10% glycerol, 2 mM DTT, 1 mM PMSF, 1x protease inhibitor (Nacalai Tesque, Inc., Kyoto, Japan)). Complexes were immunoprecipitated with 1 µg of anti-Vif mAb (#319) (a kind gift from Dr. M. Malim through the AIDS Research and Reference Reagent Program) (24) for 1 h, followed by addition of 20 µl of protein G-SepharoseTM beads (Amersham Biosciences) for 1 h at 4 °C. The beads were washed with lysing buffer three times and analyzed on immunoblot with anti-c-myc mAb (9E10) (Sigma-Aldrich Corp.) for detection of c-myc-Cul5 or rabbit anti-Vif serum.
Expression of Recombinant Proteins in HEK293T Cells—HEK293T cells were transfected with pcDNA3/HA-WT or D128K APOBEC3G by calcium phosphate method. Two days after transfection, cells were lysed in lysing buffer. Proteins were immunoprecipitated with 1 µg of anti-HA mAb (12CA5) for 1 h, followed by addition of 20 µg of protein G-SepharoseTM beads for 4 h at 4 °C. The beads were washed with lysing buffer three times and washing buffer (20 mM Tris-Cl, 10 mM DTT) twice.
Expression of Recombinant Proteins in Insect Cells—N-terminal-(His)6-tagged WT and mutants Vif were cloned into pVL1393 vectors (Invitrogen). Recombinant baculovirus was generated by using Bac-PAK6 baculovirus expression system (Clontech). N-terminal c-myc-tagged Cul5, C-terminal-HPC4-tagged EloB, C-terminal herpes simplex virus-tagged EloC, and N-terminal T7-tagged human Rbx1 were described previously (14). Hi Five cells were cultured in Grace's insect medium (Invitrogen) supplemented with 10% FCS at 27 °C and co-infected with Vif WT or mutants, Cul5, EloB, EloC, and Rbx1 recombinant viruses. Sixty-four hours after infection, cells were harvested and lysed in insect cell lysing buffer (2% Triton X-100, 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 10% glycerol, 2 mM DTT, 1 mM PMSF). Vif proteins were purified by TALON metal affinity resin (Clontech). Recombinant His-HA-APOBEC3G protein was also obtained by the same method.
In Vitro Ubiquitin Conjugation Assay—N-terminal (His)6-tagged NEDD8, Ubc12 (E2 for NEDD8) and APP-BP/Uba3 (E1 for NEDD8) were described previously (25). The plasmid encoding glutathione S-transferase (GST)-ubiquitin was kindly provided by Dr. Peter Howley (Harvard Medical School), and GST-ubiquitin was expressed and purified in our laboratory. Vif WT or mutant complexes were incubated with 4 µl of packed and washed antibody-conjugated beads containing HA-WT or D128K APOBEC3G in reaction buffer (20 mM Tris-Cl, pH 7.5, 5 mM MgCl2, 2 mM DTT, 100 ng of E1, 75 mM E2, 1 µg of NEDD8, 160 ng of Ubc12, 50 ng of APP-BP1/Uba3, 2.5 µg of GST-ubiquitin) in the presence of ATP and ATP regeneration system (1 mM ATP, 0.5 mM creatine phosphate, 5 µg of creatine phosphokinase) at 37 °C for 1 h. Reactions were stopped by adding 4x SDS sample buffer. After being boiled for 10 min, samples were subjected to Western blot to detect GST-ubiquitin conjugated HA-APOBEC3G. We also performed this assay using recombinant His-HA-APOBEC3G protein purified from insect cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Vif virions to the same extent, while the antiviral activity of WT APOBEC3G was inhibited by Vif. The amount of WT APOBEC3G protein incorporated into virions as well as in producer cells in the presence of HIV-1 Vif was much lower than that of D128K APOBEC3G (Fig. 1B), indicating that HIV-1 Vif prevented virion incorporation of WT APOBEC3G by effectively reducing its intracellular level of the protein in producer cells but not that of D128K. These results confirmed the previous reports that APOBEC3G D128K is resistant to HIV-1 Vif. Moreover, HIV-1 Vif was co-immunoprecipitated with APOBEC3G WT, but not D128K (Fig. 1C), suggesting that Vif could not down-regulate the expression of D128K because Vif cannot bind to the mutant.
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Vif virions could not restore the infectivity of the virions produced from non-permissive cells. We then tested the function of these mutants in the context of inhibition of the antiviral activity of APOBEC3G. Vif WT could overcome the antiviral activity of APOBEC3G on Vif virions, but Vif mutants not (Fig. 2A). Inability of the Vif mutants to prevent virion incorporation of APOBEC3G was attributed to their defect to down-regulate its expression in producer cells because WT Vif could reduce the amount of APOBEC3G in both virion and producer cells (Fig. 2B). Co-immunoprecipitation assays revealed that APOBEC3G could interact with all these mutants as well as WT Vif (Fig. 2C). These results indicated that interaction between APOBEC3G and Vif could not explain Vif function to antagonize APOBEC3G. We then examined whether these Vif mutants could form a complex with EloB, EloC, and Cul5 in baculovirus-infected insect cells (Fig. 2D). Lysates from Hi Five insect cells infected with Vif WT or mutants encoding baculoviruses together with EloB, EloC, and Cul5 encoding baculoviruses were immunoprecipitated with anti-Vif antibody, and the amount of Cul5 co-immunoprecipitaed with Vif was probed. An SLQ144/146AAA mutant, in which highly conserved SLQ residues required for the interaction with EloC were substituted for AAA (27), could not interact with Cul5 as reported previously (11–13), indicating that the mutant cannot inhibit APOBEC3G function because it cannot form a Vif-BC-Cul5 complex, which is critical for the down-regulation of APOBEC3G. In contrast, C114S and C133S Vif mutants could bind to Cul5 possibly through EloB and EloC, indicating that the reason why both C114S and C133S mutants could not antagonize APOBEC3G was still unclear because these mutants could interact with APOBEC3G (Fig. 2C) as well as form a Vif-BC-Cul5 complex (Fig. 2D).
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Finally, the reason why Vif C114S and C133S mutants cannot antagonize APOBEC3G was still unclear because both mutants could interact with APOBEC3G (Fig. 2C) as well as form a Vif-BC-Cul5 complex (Fig. 2D). We, therefore, tested the E3 ligase activity of the Vif C114S- or C133S-BC-Cul5 complex using this assay (Fig. 3E). The WT Vif-BC-Cul5 complex could ubiquitinate APOBEC3G effectively (lane 3), whereas the Vif C114S- or C133S-BC-Cul5 complex almost lost the E3 ligase activity toward APOBEC3G (lanes 4 and 5, respectively), although each complex contained similar level of each protein (Fig. 3F). We thus concluded that the ubiquitin ligase activity of Vif mutants through the Vif-BC-Cul5 complex is strongly correlated with their ability to inhibit APOBEC3G.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TrCP complex by binding to TrCP (29). In the latter case, adenovirus proteins E4orf6 and E1B55K form an E3 complex with BC-Cul5 to ubiquitinate p53 (30, 31). Vif acts as a substrate recognition subunit of an E3 ligase complex like adenovirus protein but most importantly to induce the ubiquitination and degradation of the antiviral host protein directly. We have then investigated the ability of several loss-of-function Vif mutants to ubiquitinate APOBEC3G using the in vitro ubiquitin conjugation assay. Vif SLQ144/146AAA mutant lost its activity against APOBEC3G because it could not form a Vif-BC-Cul5 ligase complex due to mutations in SOCS-box motif, which is critical for binding to EloC. Our findings confirm the notion revealed recently that a novel SOCS-box motif in Vif is responsible for and regulates assembly of a Vif-BC-Cul5 complex.
More importantly, we have demonstrated that both Vif C114S and C133S mutants can form a complex with BC-Cul5 (Figs. 2D and 3E) but cannot ubiquitinate APOBEC3G. Two recent reports have shown different results from ours. Mehle et al. (12) has shown that C114/133S double mutant have a weak interaction with Cul5, whereas Yu et al. (13) has shown that C114S and C133S mutants could bind to EloB-EloC complex but not to Cul5. These two reports suggest that these cysteine residues are important for assembly of a Vif-BC-Cul5 complex. However, our data clearly showed that both Vif C114S and C133S could form the Vif-BC-Cul5 complex in insect cells, and the complex could be purified. This discrepancy between the previous observations and ours cannot be fully explained at this moment, but we suspect that it is because the amount of components of Vif-BC-Cul5 is larger in baculovirus-infected insect cells than 293T cells, in which the binding experiments of the previous reports were employed. Alternatively, post-translational modification(s) to strengthen the binding between Vif-BC and Cul5 might be occurred in insect cells. Moreover, our in vitro ubiquitin conjugation assay clearly showed that purified Vif C114S- and C133S-BC-Cul5 complexes could not ubiquitinate APOBEC3G. These Vif mutants retained the ability to bind to BC-Cul5 complex as well as APOBEC3G, namely, these Vif mutants can recognize APOBEC3G as a substrate recognition subunit of a Vif-BC-Cul5 ligase. If so, why do these complexes containing Vif C114S or C133S fail to ubiquitinate APOBEC3G? The crystal structure of the SCFSkp2 ligase, a family of cullin-based ligases, has shown that the ligase has a rigid structure and the positioning between E2 and the substrate is crucial for the ligase activity (32, 33). When N-terminal domain, critical for substrate binding, and C-terminal half (E2-binding site) of cullin-1 (Cul1) were linked with a flexible linker, the ligase could not ubiquitinate the substrate, although the mutated Cul1 could bind both E2 and the substrate (32). This suggested that Cul1 ensures the fine positioning between E2 and the substrate because it is a rigid protein. Thus, in the case of Vif-BC-Cul5, the mutation of C114 or C133 may affect the conformation of Vif and perturb the position of APOBEC3G in the ligase complex, resulting in the loss of the E3 activity of the Vif-BC-Cul5 complex. However, it might be possible that stability of these complexes containing C114S or C133S is too weak to ubiquitinate APOBEC3G. The mechanism for loss of the E3 activity of C114S- and C133S-containing complexes should be fully elucidated in the future.
Finally, our results clearly suggested that loss of ubiquitin ligase activity of the Vif-BC-Cul5 complex is tightly linked to the loss of Vif function against APOBEC3G. In other words, ubiquitination of APOBEC3G by the Vif-BC-Cul5 complex is essential for Vif function. Our in vitro ubiquitin conjugation assay is a powerful tool to analyze the molecular mechanisms of ubiquitination of APOBEC3G by the Vif-BC-Cul5 complex, which will provide us with new insights into Vif function and identification of new targets for therapeutic strategy.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 81-75-751-3152; Fax: 81-75-751-4963; E-mail: atakaori{at}kuhp.kyoto-u.ac.jp.
1 The abbreviations used are: HIV-1, human immunodeficiency virus type 1; Vif, virion infectivity factor; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase enzyme; WT, wild type; HA, hemagglutinin; FCS, fetal calf serum; PSG, penicillin, streptomycin, and glutamine; mAb, monoclonal antibody; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; GST, glutathione S-transferase; NTA, nitrilotriacetic acid.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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