Key points are not available for this paper at this time.
APOBEC3G belongs to the family of cellular cytidine deaminase-editing enzymes with a potent antiretroviral activity, which is counteracted by the Vif protein expressed by lentiviruses. Antiretroviral activity of APOBEC3G requires its packaging into assembling virions, presumably to ensure its close association with nascent retroviral cDNA. Here, we demonstrate that APOBEC3G is encapsidated through a direct interaction with the HIV-1 Gag polyprotein which likely takes place on the membranes of the multivesicular bodies (MVB)/late endosomal compartments. This interaction is mediated by the Gag nucleocapsid protein NC, and the N-terminal part of NC is most critical for this interaction. Binding to the NC domain would ensure that APOBEC3G will be concentrated in the viral core of mature HIV-1, in close proximity to the reverse transcription complex. APOBEC3G belongs to the family of cellular cytidine deaminase-editing enzymes with a potent antiretroviral activity, which is counteracted by the Vif protein expressed by lentiviruses. Antiretroviral activity of APOBEC3G requires its packaging into assembling virions, presumably to ensure its close association with nascent retroviral cDNA. Here, we demonstrate that APOBEC3G is encapsidated through a direct interaction with the HIV-1 Gag polyprotein which likely takes place on the membranes of the multivesicular bodies (MVB)/late endosomal compartments. This interaction is mediated by the Gag nucleocapsid protein NC, and the N-terminal part of NC is most critical for this interaction. Binding to the NC domain would ensure that APOBEC3G will be concentrated in the viral core of mature HIV-1, in close proximity to the reverse transcription complex. Productive infection by HIV-1 1The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; GFP, green fluorescent protein; HA, hemagglutinin; RIPA, radioimmune precipitation assay; VLP, virus-like particle; MVB, multivesicular body; GM1, II3NeuAc-GgOse4Cer. requires the virus-encoded accessory protein called virion infectivity factor, Vif (1Gabuzda D.H. Lawrence K. Langhoff E. Terwilliger E. Dorfman T. Haseltine W.A. Sodroski J. J. Virol. 1992; 66: 6489-6495Crossref PubMed Google Scholar), the primary role of which is to counteract the action of a cellular antiretroviral protein APOBEC3G. APOBEC3G belongs to a family of cytidine deaminase genes that in humans includes APOBEC1, APOBEC2, seven genes (or pseudogenes) designated APOBEC3A to 3G and the activation-induced deaminase (AID) gene (2Jarmuz A. Chester A. Bayliss J. Gisbourne J. Dunham I. Scott J. Navaratnam N. Genomics. 2002; 79: 285-296Crossref PubMed Scopus (590) Google Scholar, 3Wedekind J.E. Dance G.S.C. Sowden M.P. Smith H.C. Trends Genet. 2003; 19: 207-216Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Recently another member of the APOBEC family, APOBEC3F, was shown to inhibit HIV-1 replication by a mechanism similar to that of APOBEC3G (4Zheng Y.-H. Irwin D. Kurosu T. Tokunaga K. Sata T. Peterlin B.M. J. Virol. 2004; 78: 6073-6076Crossref PubMed Scopus (392) Google Scholar). The antiretroviral activity of APOBEC3G is attributed, at least partially (5Shindo K. Takaori-Kondo A. Kobayashi M. Abudu A. Fukunaga K. Uchiyama T. J. Biol. Chem. 2003; 278: 44412-44416Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), to deamination of cytosine to uracil on the newly synthesized minus strand of viral DNA that may lead to corresponding changes of guanidines to adenines on the coding strand (6Zhang H. Yang B. Pomerantz R.J. Zhang C. Arunachalam S.C. Gao L. Nature. 2003; 424: 94-98Crossref PubMed Scopus (919) Google Scholar, 7Harris R.S. Bishop K.N. Sheehy A.M. Craig H.M. Petersen-Mahrt S.K. Watt I.N. Neuberger M.S. Malim M.H. Cell. 2003; 113: 803-809Abstract Full Text Full Text PDF PubMed Scopus (1144) Google Scholar, 8Mangeat B. Turelli P. Caron G. Friedli M. Perrin L. Trono D. Nature. 2003; 424: 99-103Crossref PubMed Scopus (1248) Google Scholar). Consequently, these mutations may result in nonfunctional provirus or degradation of the viral genome (6Zhang H. Yang B. Pomerantz R.J. Zhang C. Arunachalam S.C. Gao L. Nature. 2003; 424: 94-98Crossref PubMed Scopus (919) Google Scholar, 7Harris R.S. Bishop K.N. Sheehy A.M. Craig H.M. Petersen-Mahrt S.K. Watt I.N. Neuberger M.S. Malim M.H. Cell. 2003; 113: 803-809Abstract Full Text Full Text PDF PubMed Scopus (1144) Google Scholar, 8Mangeat B. Turelli P. Caron G. Friedli M. Perrin L. Trono D. Nature. 2003; 424: 99-103Crossref PubMed Scopus (1248) Google Scholar). Analysis of the mutations generated by APOBEC3G in HIV-1 DNA showed that single DNA strand specificity of APOBEC3G is responsible for deamination of the HIV DNA minus strand (9Yu Q. Konig R. Pillai S. Chiles K. Kearney M. Palmer S. Richman D. Coffin J.M. Landau N.R. Nat. Struct. Mol. Biol. 2004; 11: 435-442Crossref PubMed Scopus (506) Google Scholar). Antiretroviral activity of APOBEC3G requires its encapsidation into assembling virions, presumably to ensure its close association with the retroviral reverse transcription complex and nascent retroviral cDNA. In the absence of Vif, APOBEC3G is specifically incorporated into the assembling HIV-1 virions, reducing infectivity of the newly produced virus (7Harris R.S. Bishop K.N. Sheehy A.M. Craig H.M. Petersen-Mahrt S.K. Watt I.N. Neuberger M.S. Malim M.H. Cell. 2003; 113: 803-809Abstract Full Text Full Text PDF PubMed Scopus (1144) Google Scholar, 10Mariani R. Chen D. Schrofelbauer B. Navarro F. Konig R. Bollman B. Munk C. Nymark-McMahon H. Landau N.R. Cell. 2003; 114: 21-31Abstract Full Text Full Text PDF PubMed Scopus (771) Google Scholar, 11Liu B. Yu X. Luo K. Yu Y. Yu X.-F. J. Virol. 2004; 78: 2072-2081Crossref PubMed Scopus (131) Google Scholar). However, expression of HIV-1 Vif reduces incorporation of APOBEC3G into virions (10Mariani R. Chen D. Schrofelbauer B. Navarro F. Konig R. Bollman B. Munk C. Nymark-McMahon H. Landau N.R. Cell. 2003; 114: 21-31Abstract Full Text Full Text PDF PubMed Scopus (771) Google Scholar, 12Kao S. Khan M.A. Miyagi E. Plishka R. Buckler-White A. Strebel K. J. Virol. 2003; 77: 11398-11407Crossref PubMed Scopus (269) Google Scholar, 13Marin M. Rose K.M. Kozak S.L. Kabat D. Nat. Med. 2003; 9: 1398-1403Crossref PubMed Scopus (683) Google Scholar, 14Sheehy A.M. Gaddis N.C. Choi J.D. Malim M.H. Nature. 2002; 418: 646-650Crossref PubMed Scopus (1914) Google Scholar, 15Stopak K. de Noronha C. Yonemoto W. Greene W.C. Mol. Cell. 2003; 12: 591-601Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, 16Yu X. Yu Y. Liu B. Luo K. Kong W. Mao P. Yu X.-F. Science. 2003; 302: 1056-1060Crossref PubMed Scopus (1002) Google Scholar), possibly by binding to APOBEC3G and promoting its degradation via the ubiquitin-proteasome pathway (13Marin M. Rose K.M. Kozak S.L. Kabat D. Nat. Med. 2003; 9: 1398-1403Crossref PubMed Scopus (683) Google Scholar, 15Stopak K. de Noronha C. Yonemoto W. Greene W.C. Mol. Cell. 2003; 12: 591-601Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, 17Conticello S.G. Harris R.S. Neuberger M.S. Curr. Biol. 2003; 13: 2009-2013Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 18Mehle A. Strack B. Ancuta P. Zhang C. McPike M. Gabuzda D. J. Biol. Chem. 2004; 279: 7792-7798Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 19Sheehy A.M. Gaddis N.C. Malim M.H. Nat. Med. 2003; 9: 1404-1407Crossref PubMed Scopus (809) Google Scholar). The ability of Vif to block the antiviral activity of APOBEC3G is species-specific (10Mariani R. Chen D. Schrofelbauer B. Navarro F. Konig R. Bollman B. Munk C. Nymark-McMahon H. Landau N.R. Cell. 2003; 114: 21-31Abstract Full Text Full Text PDF PubMed Scopus (771) Google Scholar), and a region of APOBEC3G determining this specificity has been localized to a single amino acid residue (19Sheehy A.M. Gaddis N.C. Malim M.H. Nat. Med. 2003; 9: 1404-1407Crossref PubMed Scopus (809) Google Scholar, 20Schrofelbauer B. Chen D. Landau N.R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3927-3932Crossref PubMed Scopus (285) Google Scholar, 21Bogerd H.P. Doehle B.P. Wiegand H.L. Cullen B.R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3770-3774Crossref PubMed Scopus (276) Google Scholar, 22Mangeat B. Turelli P. Liao S. Trono D. J. Biol. Chem. 2004; 279: 14481-14483Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 23Xu H. Svarovskaia E.S. Barr R. Zhang Y. Khan M.A. Strebel K. Pathak V.K. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5652-5657Crossref PubMed Scopus (223) Google Scholar). The mechanism by which APOBEC3G is incorporated into assembling virions is unknown. Analysis of the molecular mechanism(s) by which this antiviral protein is targeted to virus assembly sites may suggest an alternative approach to increase its virion levels in the presence of Vif. The precise cellular distribution of APOBEC3G has not yet been investigated but may provide an insight into the mechanism of its incorporation into virions. Results presented herein suggest that APOBEC3G is encapsidated through a direct interaction with the nucleocapsid of HIV-1 Gag. Moreover, we suggest that binding of APOBEC3G to abundantly produced Gag during infection may lead not only to its encapsidation but also result in degradation of APOBEC3G complexed with Gag. Both mechanisms may significantly deplete APOBEC3G levels independently of Vif. Reagents—Recombinant APOBEC3G and anti-APOBEC3G monoclonal antibodies were from ImmunoDiagnostics. Mouse anti-GFP 3E6, rabbit anti-GFP, LysoTracker Red, and transferrin Alexa Fluor 594 were from Molecular Probes. Mouse anti-α-tubulin, anti-CD63/LAMP-3, anti-Rab7 antibodies, and protein A/G plus-agarose were from Santa Cruz Biotechnology. Anti-HA, mouse monoclonal 12CA5 and rat monoclonal 3F10 antibodies were from Roche Applied Science. Mouse anti-CD71 was from Beckman Coulter. Mouse anti-His and anti-EEA1 antibodies were from Invitrogen and BD Transduction Laboratories, respectively. Virion Encapsidation Assay—4 or 10 ml of culture supernatant from transfected 293T cells were filtered through 0.45-μm filters and concentrated by ultracentrifugation in a SW 60 or SW 41 rotor (Beckman) at 25,000 rpm for 2 h through a layer of 20% sucrose in phosphate-buffered saline. The pelleted virus-like particles were solubilized in 50–100 μl of RIPA buffer (50 mm Tris, pH 7.5, 10 mm EDTA, 150 mm NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with protease inhibitors (Complete; Roche Applied Science), and 40 μl was analyzed by Western blotting. Western Blotting and Immunoprecipitation—Western blot analysis was performed as described (24Popik W. Alce T.M. J. Biol. Chem. 2004; 279: 704-712Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). For immunoprecipitation, postnuclear cell lysates in RIPA buffer were prepared from transfected 293T cells. Cell lysates (0.5–1 mg of protein) were immunoprecipitated overnight with anti-GFP 3E6 monoclonal antibody and protein A/G plus-agarose. After extensive washing, the precipitates were subjected to SDS-PAGE and analyzed by Western blotting. In Vitro Binding Experiments—HIV-1 Gag and various Gag mutants (constructed using the QuikChange site-directed mutagenesis kit; Stratagene) were expressed as GST fusion proteins in Escherichia coli and were purified from bacterial lysates using glutathione-Sepharose beads (25Burniston M.T. Cimarelli A. Colgan J. Curtis S.P. Luban J. J. Virol. 1999; 73: 8527-8540Crossref PubMed Google Scholar). Recovery of the GST and GST-Gag fusion proteins was monitored by staining the gels with Coomassie Blue. The glutathione-Sepharose beads with associated GST (negative control) or GST-Gag proteins were incubated overnight at 4 °C with recombinant APOBEC3G or with cell lysates prepared from transfected 293T cells expressing APOBEC3G tagged with GFP, His, or HA. After washing, bound proteins were eluted by boiling in SDS Laemmli buffer, resolved by SDS-PAGE, and visualized by Western blotting. Confocal Microscopy—293T cells were grown on glass chamber slides (Nalgene Nunc International) and transfected with GFP-APOBEC3G and Gag or Gag-GFP expression vectors, and 24 h later, the cells were fixed with 4% paraformaldehyde, washed, and analyzed by confocal microscopy (24Popik W. Alce T.M. J. Biol. Chem. 2004; 279: 704-712Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). For labeling of the early or late endosomes, the transfected cells were incubated for 30 min at 37 °C with transferrin conjugated with Alexa Fluor 594 or with LysoTracker Red, respectively (Molecular Probes), according to the manufacturer's protocols. The images were taken with a LSM 510 META confocal microscope (Zeiss). Biochemical Analysis of APOBEC3G Binding to Cellular Membranes and Lipid Rafts—Transfected 293T cells were harvested in 20 mm Tris, pH 7.5, 1 mm EDTA buffer supplemented with protease inhibitors and disrupted by Dounce homogenization. The postnuclear supernatant was separated into total membrane and cytosolic fractions by centrifugation at 40,000 × g for 30 min (26Sciorra V.A. Rudge S.A. Prestwich G.D. Frohman M.A. Engebrecht J. Morris A.J. EMBO J. 1999; 20: 5911-5921Crossref Scopus (147) Google Scholar). Endosomal membranes were prepared from transfected cells by centrifugation of the postnuclear supernatant on a discontinuous sucrose gradient (27Sun W. Yan Q. Vida T.A. Bean A.J. J. Cell Biol. 2003; 162: 125-137Crossref PubMed Scopus (61) Google Scholar). Lipid rafts were prepared using 0.5% Triton X-100 as described in detail (24Popik W. Alce T.M. J. Biol. Chem. 2004; 279: 704-712Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 28Popik W. Alce T.M. Au W.-C. J. Virol. 2002; 76: 4709-4722Crossref PubMed Scopus (285) Google Scholar). Transfection of GFP-APOBEC3G with increasing amounts of HIV-1 Gag reduced intracellular GFP-APOBEC3G steady-state levels in a concentration-dependent manner (Fig. 1A). Since Gag itself is sufficient to drive the assembly of virus-like particles (VLPs), we reasoned that reduction of GFP-APOBEC3G expression in cells resulted from APOBEC3G packaging into VLPs, most likely through the interaction with Gag. To test this hypothesis, 293T cells were transfected, and 24 h later culture supernatants were collected, filtered, and ultracentrifuged to pellet VLPs. Cell lysates were prepared from the same transfected cells, and both VLPs and lysates were analyzed by Western blotting for the presence of Gag and GFP-APOBEC3G (Fig. 1B). The results show that decreasing steady-state levels of GFP-APOBEC3G in cell lysates correlated with the increasing amount of transfected Gag expression vector. This increased amount of Gag resulted in greater accumulation of APOBEC3G in VLPs. Moreover, in the presence of Gag, GFP-APOBEC3G redistributed into a discrete punctate pattern localized close to, or at, the cell surface (Fig. 2A) and most likely represents coaggregating Gag and APOBEC3G (Fig. 2B). These results suggest that APOBEC3G interacts with Gag in the absence of other viral proteins. This possibility was further substantiated by the observation that Gag and APOBEC3G were bound to cell membranes pelleted from postnuclear supernatants prepared from transfected 293T cells in the absence of detergent (Fig. 2C). Interestingly, APOBEC3G localized to Triton X-100-resistant lipid rafts (Fig. 2D), which probably are not of plasma membrane origin, since APOBEC3G did not noticeably localize to the plasma membrane (data not shown). Thus, we hypothesize that APOBEC3G associates with intracellular membrane rafts. Since Gag concentrates both in the plasma membrane and multivesicular bodies (MVB)/late endosomal compartments (29Pelchen-Matthews A. Kramer B. Marsh M. J. Cell Biol. 2003; 162: 443-455Crossref PubMed Scopus (400) Google Scholar, 30Nydegger S. Foti M. Derdowski A. Spearman P. Thali M. Traffic. 2003; 4: 902-910Crossref PubMed Scopus (150) Google Scholar, 31Ono A. Freed E.O. J. Virol. 2004; 78: 1552-1563Crossref PubMed Scopus (228) Google Scholar) enriched in lipid rafts and both Gag and APOBEC3G colocalize (Fig. 2B), we speculate that APOBEC3G may interact with Gag at the membranes of the MVB/late endosomal compartments. Indeed, APOBEC3G partially cofractionated with the late endosomal/lysosomal markers, Rab7 and CD63/LAMP-3 (Fig. 3A). Solubilization of APOBEC3G as well as Rab7 and CD63 with the mild nonionic detergent octyl glucoside shows that these proteins were indeed associated with membrane fractions. Coexpression of Gag-GFP and APOBEC3G-HA in 293T cells resulted in significant accumulation of APOBEC3G in Rab7-positive and EEA1-negative late endosomes (Fig. 3B), suggesting that accumulation of Gag on MVB/late endosomes may be responsible for bringing more APOBEC3G to this compartment. These results were confirmed by confocal analysis using fluorescent LysoTracker Red, a specific probe for labeling late endosomes and lysosomes (Fig. 3C). While Gag-GFP and GFP-APOBEC3G colocalized with the LysoTracker-labeled compartment, significantly less colocalization was observed between APOBEC3G and the early endosomal marker transferrin-Alexa Fluor 594, suggesting that APOBEC3G was not associated with intracellular membranes but specifically targeted to late endosomal these the that APOBEC3G and Gag interact on intracellular most likely in the late endosomal colocalization of APOBEC3G and and images that in the presence of Gag, GFP-APOBEC3G into a discrete punctate pattern colocalization of GFP-APOBEC3G with in Gag and GFP-APOBEC3G to cell membranes pelleted from postnuclear supernatant prepared from transfected 293T cells. Western blot analysis shows that Gag and APOBEC3G to cytosolic and fractions and APOBEC3G to lipid rafts independently of Gag. lysates in 0.5% Triton X-100 were prepared from transfected 293T cells and on sucrose fractions were resolved by SDS-PAGE and with and anti-GFP antibodies to APOBEC3G and Gag, respectively. of and marker is of GFP-APOBEC3G and endosomal APOBEC3G with late endosomal membrane 293T cells were disrupted in buffer 20 mm pH 2 mm supplemented with protease The postnuclear supernatants were into part was the other was incubated with 0.5% nonionic detergent were to sucrose and with and sucrose and The gradient was for 1 h at × were from the of the gradient and were analyzed by Western blotting for the presence of APOBEC3G and endosomal markers, Rab7 and GFP-APOBEC3G and Gag-GFP colocalize with Rab7-positive and EEA1-negative late were with Gag-GFP and APOBEC3G-HA and postnuclear supernatants were prepared and as described in were and designated as the membrane early endosomes and late endosomes as described T. E. de P. J. Nature. PubMed Scopus Google Scholar). of postnuclear were analyzed by Western blotting for the presence of Gag, Rab7 and colocalization of GFP-APOBEC3G and Gag-GFP with late endosomal/lysosomal marker LysoTracker cells were incubated for 30 min at 37 °C with fluorescent a specific probe for labeling of late endosomes and lysosomes or with the early endosomal marker transferrin-Alexa Fluor 594 and as described images of GFP-APOBEC3G or Gag-GFP with LysoTracker or transferrin-Alexa Fluor 594 are To test the interaction between Gag and in binding were performed with recombinant proteins expressed in E. HIV-1 Gag was expressed as a GST-Gag fusion on glutathione-Sepharose beads (Fig. Coomassie and and for the ability to recombinant APOBEC3G or prepared from lysates of transfected 293T cells (Fig. Results show that APOBEC3G associated with GST-Gag but not with that the interaction was specific for Gag. to the Gag domain that interacts with Gag mutants expressing or were produced as GST fusion proteins (Fig. Coomassie and for the ability to APOBEC3G. The results demonstrate that in the absence of the NC domain is binding to APOBEC3G. Thus, the Gag NC domain is likely to the interaction with APOBEC3G. Since the of recombinant APOBEC3G and GST-Gag was greater that proteins as for the association between Gag and APOBEC3G in this To that the interaction between Gag and APOBEC3G in a was performed (Fig. 293T cells were transfected with expression for Gag-GFP or Gag-GFP mutants and 24 h later, the cellular lysates were immunoprecipitated with monoclonal anti-GFP proteins were analyzed by Western blotting for the presence of Gag and APOBEC3G using specific Results of the show that APOBEC3G was immunoprecipitated with anti-GFP antibodies only with Gag-GFP and in the presence of Gag NC domain (Fig. confirmed the of Gag NC domain in GST The of the NC domain in the interaction with APOBEC3G was further confirmed by that the NC domain with GST was sufficient to interact in with APOBEC3G (Fig. However, in binding of APOBEC3G to the domain was not as as in in binding This may be to the by anti-GFP antibodies binding in close proximity to NC in the complex. Interestingly, this was S. F. M. J. J. L. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) showed that HIV-1 Gag is sufficient for packaging of APOBEC3G into Gag VLPs, and this incorporation requires the presence of Gag Thus, these HIV-1 Gag NC is in and To more which region of NC is responsible for the observed interaction with mutants were produced as GST fusion proteins. The amino acid were in NC and in NC Both in GST fusion (Fig. and (Fig. show that the N-terminal domain of NC acid is critical for the interaction with the NC domain also partially to the binding with APOBEC3G. results demonstrate that the antiretroviral protein APOBEC3G is incorporated into viral particles by direct binding to the NC domain of the Gag hypothesize that the interaction with the NC domain that APOBEC3G will be to the HIV-1 core during virus and HIV-1 and reverse and will to the reverse transcription produced virus results also the possibility that of Gag proteins observed during infection which only a will be incorporated into virions, the most likely may as a for APOBEC3G and deplete the cellular to levels for encapsidation and HIV-1 DNA of these results is that of the interaction between APOBEC3G and Vif by may APOBEC3G may not be sufficient to of APOBEC3G by HIV-1 of HIV-1 Gag NC to the interaction with Gag NC mutants the of NC amino or of NC amino as well as the NC domain were produced and purified as GST fusion proteins Coomassie and for the ability to GFP-APOBEC3G expressed in transfected 293T cells. GFP-APOBEC3G was visualized by Western blotting with anti-GFP antibodies cell lysates and VLPs prepared from transfected were analyzed for the presence of Gag-GFP mutants and APOBEC3G by Western blotting. of Gag-GFP NC mutants and Gag with APOBEC3G-HA expressed in 293T cells was performed as described in the to that since in and anti-GFP is the other Gag-GFP proteins for is not shown according to its molecular A. M. M. H. and L. for the of
Alce et al. (Thu,) studied this question.