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Human Apolipoprotein B mRNA-editing Enzyme-catalytic Polypeptide-like 3G (APOBEC3G) Is Incorporated into HIV-1 Virions through Interactions with Viral and Nonviral RNAs

2004; Elsevier BV; Volume: 279; Issue: 34 Linguagem: Inglês

10.1074/jbc.m405761200

ISSN

1083-351X

Autores

Evguenia S. Svarovskaia, Hongzhan Xu, Jean L. Mbisa, Rebekah Barr, Robert J. Gorelick, Akira Ono, Eric O. Freed, Wei-Shau Hu, Vinay K. Pathak,

Tópico(s)

HIV/AIDS drug development and treatment

Resumo

Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is a host cytidine deaminase that is packaged into virions and confers resistance to retroviral infection. APOBEC3G deaminates deoxycytidines in minus strand DNA to deoxyuridines, resulting in G to A hypermutation and viral inactivation. Human immunodeficiency virus type 1 (HIV-1) virion infectivity factor counteracts the antiviral activity of APOBEC3G by inducing its proteosomal degradation and preventing virion incorporation. To elucidate the mechanism of viral suppression by APOBEC3G, we developed a sensitive cytidine deamination assay and analyzed APOBEC3G virion incorporation in a series of HIV-1 deletion mutants. Virus-like particles derived from constructs in which pol, env, and most of gag were deleted still contained high levels of cytidine deaminase activity; in addition, coimmunoprecipitation of APOBEC3G and HIV-1 Gag in the presence and absence of RNase A indicated that the two proteins do not interact directly but form an RNase-sensitive complex. Viral particles lacking HIV-1 genomic RNA which were generated from the gag-pol expression constructs pC-Help and pSYNGP packaged APOBEC3G at 30–40% of the wild-type level, indicating that interactions with viral RNA are not necessary for incorporation. In addition, viral particles produced from an nucleocapsid zinc finger mutant contained ∼1% of the viral genomic RNA but ∼30% of the cytidine deaminase activity. The reduction in APOBEC3G incorporation was equivalent to the reduction in the total RNA present in the nucleocapsid mutant virions. These results indicate that interactions with viral proteins or viral genomic RNA are not essential for APOBEC3G incorporation and suggest that APOBEC3G interactions with viral and nonviral RNAs that are packaged into viral particles are sufficient for APOBEC3G virion incorporation. Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is a host cytidine deaminase that is packaged into virions and confers resistance to retroviral infection. APOBEC3G deaminates deoxycytidines in minus strand DNA to deoxyuridines, resulting in G to A hypermutation and viral inactivation. Human immunodeficiency virus type 1 (HIV-1) virion infectivity factor counteracts the antiviral activity of APOBEC3G by inducing its proteosomal degradation and preventing virion incorporation. To elucidate the mechanism of viral suppression by APOBEC3G, we developed a sensitive cytidine deamination assay and analyzed APOBEC3G virion incorporation in a series of HIV-1 deletion mutants. Virus-like particles derived from constructs in which pol, env, and most of gag were deleted still contained high levels of cytidine deaminase activity; in addition, coimmunoprecipitation of APOBEC3G and HIV-1 Gag in the presence and absence of RNase A indicated that the two proteins do not interact directly but form an RNase-sensitive complex. Viral particles lacking HIV-1 genomic RNA which were generated from the gag-pol expression constructs pC-Help and pSYNGP packaged APOBEC3G at 30–40% of the wild-type level, indicating that interactions with viral RNA are not necessary for incorporation. In addition, viral particles produced from an nucleocapsid zinc finger mutant contained ∼1% of the viral genomic RNA but ∼30% of the cytidine deaminase activity. The reduction in APOBEC3G incorporation was equivalent to the reduction in the total RNA present in the nucleocapsid mutant virions. These results indicate that interactions with viral proteins or viral genomic RNA are not essential for APOBEC3G incorporation and suggest that APOBEC3G interactions with viral and nonviral RNAs that are packaged into viral particles are sufficient for APOBEC3G virion incorporation. Retroviral genomes, including that of HIV-1, 1The abbreviations used are: HIV-1, human immunodeficiency virus type 1; APOBEC3G, apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like-3G; CA, capsid; EGFP, enhanced green fluorescent protein; eIF3k, cytoplasmic translational initiation factor 3 subunit k; MA, matrix; NC, nucleocapsid; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; Ψ, HIV-1 packaging signal; Vif, virion infectivity factor; VLP, virus-like particle; Gag, group antigen; HDV, HIV-1-derived vector.1The abbreviations used are: HIV-1, human immunodeficiency virus type 1; APOBEC3G, apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like-3G; CA, capsid; EGFP, enhanced green fluorescent protein; eIF3k, cytoplasmic translational initiation factor 3 subunit k; MA, matrix; NC, nucleocapsid; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; Ψ, HIV-1 packaging signal; Vif, virion infectivity factor; VLP, virus-like particle; Gag, group antigen; HDV, HIV-1-derived vector. undergo massive G to A substitutions in one or a few rounds of replication (1Pathak V.K. Temin H.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6019-6023Crossref PubMed Scopus (217) Google Scholar, 2Vartanian J.P. Meyerhans A. Asjo B. Wain-Hobson S. J. Virol. 1991; 65: 1779-1788Crossref PubMed Google Scholar). Because this high rate of G to A substitution (∼2 mutations/100 nucleotides) is 1,000-fold greater than the rate of substitutions that arise during normal error-prone viral replication (∼2 × 10-5 mutations/nucleotides/replication), it is named hypermutation (1Pathak V.K. Temin H.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6019-6023Crossref PubMed Scopus (217) Google Scholar). Recent studies indicate that the HIV-1 Vif protein protects viral replication from a host restriction factor that induces hypermutation of the HIV-1 genome (3Lecossier D. Bouchonnet F. Clavel F. Hance A.J. Science. 2003; 300: 1112Crossref PubMed Scopus (587) Google Scholar, 4Mangeat B. Turelli P. Caron G. Friedli M. Perrin L. Trono D. 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Nature. 2002; 418: 646-650Crossref PubMed Scopus (1912) Google Scholar), is a dominant acting host factor that is responsible for the restricted cell phenotype (3Lecossier D. Bouchonnet F. Clavel F. Hance A.J. Science. 2003; 300: 1112Crossref PubMed Scopus (587) Google Scholar, 4Mangeat B. Turelli P. Caron G. Friedli M. Perrin L. Trono D. Nature. 2003; 424: 99-103Crossref PubMed Scopus (1245) Google Scholar, 5Zhang H. Yang B. Pomerantz R.J. Zhang C. Arunachalam S.C. Gao L. Nature. 2003; 424: 94-98Crossref PubMed Scopus (919) Google Scholar, 6Harris 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 (1139) Google Scholar, 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 (505) Google Scholar). APOBEC3G is a member of a family of cytidine deaminases that play a central role in generating somatic hypermutation and mRNA editing (10Wedekind J.E. Dance G.S. Sowden M.P. Smith H.C. Trends Genet. 2003; 19: 207-216Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Recent studies have shown that APOBEC3G expression in viral producer cells causes deamination of deoxycytidines in minus strand viral DNA to deoxyuridines, thereby resulting in G to A hypermutation (3Lecossier D. Bouchonnet F. Clavel F. Hance A.J. Science. 2003; 300: 1112Crossref PubMed Scopus (587) Google Scholar, 4Mangeat B. Turelli P. Caron G. Friedli M. Perrin L. Trono D. Nature. 2003; 424: 99-103Crossref PubMed Scopus (1245) Google Scholar, 5Zhang H. Yang B. Pomerantz R.J. Zhang C. Arunachalam S.C. Gao L. Nature. 2003; 424: 94-98Crossref PubMed Scopus (919) Google Scholar, 6Harris 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 (1139) Google Scholar, 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 (505) Google Scholar). APOBEC3G must be packaged into virions to exhibit antiviral activity because its expression in target cells does not result in hypermutation or inhibition of viral replication. HIV-1 Vif overcomes the antiviral activity of APOBEC3G by inducing its rapid degradation through the proteosomal pathway, thereby preventing its incorporation into virion (11Kao S. Khan M.A. Miyagi E. Plishka R. Buckler-White A. Strebel K. J. Virol. 2003; 77: 11398-11407Crossref PubMed Scopus (268) Google Scholar, 12Marin M. Rose K.M. Kozak S.L. Kabat D. Nat. Med. 2003; 9: 1398-1403Crossref PubMed Scopus (683) Google Scholar, 13Xu 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, 14Yu X. Yu Y. Liu B. Luo K. Kong W. Mao P. Yu X.F. Science. 2003; 302: 1056-1060Crossref PubMed Scopus (999) Google Scholar, 15Mehle 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). It has also been reported that HIV-1 Vif reduces the efficiency of translation of APOBEC3G mRNA (16Stopak K. de Noronha C. Yonemoto W. Greene W.C. Mol. Cell. 2003; 12: 591-601Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar). We and others have shown recently that a single amino acid substitution in human APOBEC3G confers resistance to HIV-1 Vif (13Xu 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, 17Mangeat B. Turelli P. Liao S. Trono D. J. Biol. Chem. 2004; 279: 14481-14483Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 18Bogerd 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, 19Schrofelbauer B. Chen D. Landau N.R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3927-3932Crossref PubMed Scopus (284) Google Scholar). The mechanism by which APOBEC3G is incorporated into HIV-1 virions as well as other diverse retroviruses such as simian immunodeficiency virus, murine leukemia virus, and equine infectious anemia virus is unknown (4Mangeat B. Turelli P. Caron G. Friedli M. Perrin L. Trono D. Nature. 2003; 424: 99-103Crossref PubMed Scopus (1245) Google Scholar, 6Harris 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 (1139) Google Scholar, 20Mariani 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 (770) Google Scholar). The proteins and RNA components of these viruses share few common determinants that could be exploited by APOBEC3G to gain entry into viral particles. Furthermore, the high evolutionary potential of HIV-1 and other retroviruses should allow them to modify potential binding determinants quickly to prevent APOBEC3G incorporation. Instead, retroviruses have evolved the elaborate mechanism of encoding Vif to induce the proteosomal degradation of APOBEC3G. On the other hand, some protein determinants, such as the YXDD catalytic domain of reverse transcriptases or the zinc finger motifs of NC proteins, are highly conserved in the orthoretroviruses and are essential for retroviral replication. Thus, these elements could potentially provide binding interactions that allow incorporation of APOBEC3G. We have now developed a novel cytidine deaminase assay to determine APOBEC3G activity in virions. Deletion analysis of HIV-1 gag-pol expression constructs and their RNA content indicates that APOBEC3G incorporation does not require a specific interaction with viral proteins or viral RNA. The results suggest that APOBEC3G is associated with viral and cellular RNAs that are specifically and nonspecifically packaged into HIV-1 virions, respectively. Plasmids—pHDV-EGFP (kindly provided by D. Unutmaz, Vanderbilt University) (21Unutmaz D. KewalRamani V.N. Marmon S. Littman D.R. J. Exp. Med. 1999; 189: 1735-1746Crossref PubMed Scopus (354) Google Scholar), pNL4-3 (22Adachi A. Gendelman H.E. Koenig S. Folks T. Willey R. Rabson A. Martin M.A. J. Virol. 1986; 59: 284-291Crossref PubMed Google Scholar), pcDNA-APO3G (11Kao S. Khan M.A. Miyagi E. Plishka R. Buckler-White A. Strebel K. J. Virol. 2003; 77: 11398-11407Crossref PubMed Scopus (268) Google Scholar), a D128K mutant of APOBEC3G (13Xu 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), pC-Help (23Mochizuki H. Schwartz J.P. Tanaka K. Brady R.O. Reiser J. J. Virol. 1998; 72: 8873-8883Crossref PubMed Google Scholar), pC-HelpΔVif (kindly provided by K. Strebel, NIAID, National Institutes of Health) (13Xu 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), and pΔNC (kindly provided by D. Ott, AIDS Vaccine Program, SAIC-Frederick) (24Ott D.E. Coren L.V. Chertova E.N. Gagliardi T.D. Nagashima K. Sowder Jr., R.C. Poon D.T. Gorelick R.J. J. Virol. 2003; 77: 5547-5556Crossref PubMed Scopus (49) Google Scholar), have been described previously. pGag expressed a full-length Gag protein that contained a FLAG tag at the C terminus. pGag, p43*, p41*, and pCA146* have been described previously (25Ono A. Demirov D. Freed E.O. J. Virol. 2000; 74: 5142-5150Crossref PubMed Scopus (104) Google Scholar, 26Ono A. Orenstein J.M. Freed E.O. J. Virol. 2000; 74: 2855-2866Crossref PubMed Scopus (199) Google Scholar). pSYNGP, an HIV-1 codon-optimized gag-pol expression construct, was obtained from Oxford Biomedica (UK) (27Kotsopoulou E. Kim V.N. Kingsman A.J. Kingsman S.M. Mitrophanous K.A. J. Virol. 2000; 74: 4839-4852Crossref PubMed Scopus (200) Google Scholar). The ΔMA construct was generated by complete deletion of MA from pNL4-3 and insertion of the first 10 amino acids of Fyn at the N terminus of gag. pMA14CA129 was generated by an in-frame deletion in pC-Help that resulted in deletion from residue 15 of MA to residue 129 in CA and a Y130R substitution in CA. pHIV-Thy1 and pHIVThy1-CCHH are similar to pHDV-EGFP except that the eGFP gene is replaced with mouse thy-1 gene. pHIV-Thy1-CCHH was constructed by replacing an SphI–SbfI fragment of HIV-Thy1 with an equivalent fragment from HIV-NL43-CCHH/CCCC containing the zinc finger mutation in NC (28Gorelick R.J. Gagliardi T.D. Bosche W.J. Wiltrout T.A. Coren L.V. Chabot D.J. Lifson J.D. Henderson L.E. Arthur L.O. Virology. 1999; 256: 92-104Crossref PubMed Scopus (122) Google Scholar). Cells, Transfections, and Virus Production—293T, HUT78, and CEM-SS were maintained as described previously (29Svarovskaia E.S. Barr R. Zhang X. Pais G.C. Marchand C. Pommier Y. Burke Jr., T.R. Pathak V.K. J. Virol. 2004; 78: 3210-3222Crossref PubMed Scopus (96) Google Scholar). CEMx174 cells were maintained in conditions identical to those used for CEM-SS cells. Peripheral blood mononuclear cells (PBMCs) and macrophages were provided by J. Chen. PBMCs were isolated from healthy donors through Histopaque gradients (Sigma), activated by 2 μg/ml phytohemagglutinin for 3 days, and maintained in RPMI medium containing 10% fetal bovine serum and 200 units/ml recombinant interleukin-2 for 3–4 days. Macrophages were grown from elutriated monocytes on non-tissue culture-treated plates in RPMI medium plus 10% human AB serum for 7 days. VLPs were produced from 293T cells transfected with HIV-1, pcDNA-APO3G encoding APOBEC3G or its D128K mutant, or both HIV-1- and APOBEC3G-expressing plasmids by using a CalPhos Mammalian Transfection Kit (BD Biosciences), harvested 24–30 h after transfections, filtered using 0.45-μm membranes (Nalgene), and concentrated 20-fold by ultracentrifugation as described previously (29Svarovskaia E.S. Barr R. Zhang X. Pais G.C. Marchand C. Pommier Y. Burke Jr., T.R. Pathak V.K. J. Virol. 2004; 78: 3210-3222Crossref PubMed Scopus (96) Google Scholar). The HIV-1 Vif-resistant mutant D128K (13Xu 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) was used to analyze APOBEC3G virion incorporation in HIV-1 gag-pol expression constructs that express HIV-1 Vif. Cytoplasmic Protein Isolation—Cytoplasmic protein extracts were prepared as described previously (29Svarovskaia E.S. Barr R. Zhang X. Pais G.C. Marchand C. Pommier Y. Burke Jr., T.R. Pathak V.K. J. Virol. 2004; 78: 3210-3222Crossref PubMed Scopus (96) Google Scholar). The total protein concentrations were determined using the Bradford protein assay (Bio-Rad Laboratories), and 1.5 μg of total cytoplasmic protein was used to determine cytidine deamination activity. Scintillation Proximity Assay for Cytidine Deamination—The sequence of the primer oligonucleotide was 5′-biotin-GTCAGCATCCTGAATTCTACC-3′. The sequence of the template oligonucleotide (CCCA)10 was 5′-ACCCACCCACCCACCCACCCACCCACCCACCCACCCACCCGGTAGAATTCAGGATGCTGAC-3′. The biotinylated primer and (CCCA)10 template oligonucleotides were annealed to each other and immobilized on streptavidin-polyvinyltoluene SPA beads (Amersham Biosciences) as recommended by the manufacturer. The cytidine deamination reaction was performed in a final volume of 100 μl for 2 h (unless otherwise specified) at 37 °C in the presence of the (CCCA)10 SPA beads in assay buffer (50 mm Tris-HCl, pH 8.0, 80 mm KCl, 10 mm MgCl2, 10 mm dithiothreitol, 2.5 mm EGTA, 0.05% (w/v) Nonidet P-40). The polymerization reaction was performed in 0.04 mm each dTTP and dGTP, 1 μCi of [3H]dATP (specific activity, 20–50 Ci/mmol; Amersham Biosciences), and 0.2 unit of Klenow (Roche Applied Science) for 20 min and stopped by the addition 200 μl of EDTA (120 mm, pH 8.0). The reaction products were diluted to 3 ml with 1× Tris borate-EDTA buffer, and the incorporation of tritiated deoxynucleotides was quantified using the Tricarb 1600TR (Packard) liquid scintillation analyzer. A parallel reaction was performed for each sample in the presence of dATP and dGTP (final concentration, 0.04 mm) and 1 μCi of [3H]dTTP (specific activity, 40–80 Ci/mmol; ICN) to control for polymerization efficiency. In addition, a (UUUA)10 oligonucleotide template, which was identical to the (CCCA)10 template except that the deoxycytidines were replaced with deoxyuridines, was used in polymerization reactions, and the resulting incorporation of [3H]dATP was referred to as 100% APO activity. Sequences of (CCA)13 and (CCCCA)8 template oligonucleotides were similar to the (CCCA)10 template and contained the indicated sequences that were repeated 13 or 8 times, respectively. To calculate APO activity for these templates, normalization to a 100% activity was adjusted according to the number of deoxycytidines in each template oligonucleotide. RNA Isolation and Quantification—To quantify the HIV-1 viral RNA content of the HIV-Thy wild-type and CCHH mutant virions, total RNA was isolated as described previously (30Fu W. Hu W.S. J. Virol. 2003; 77: 754-761Crossref PubMed Scopus (13) Google Scholar). Briefly, 500 μl of concentrated VLPs in PBS were treated with 15 units of RNase-free DNase I (Roche Applied Science) for 40 min in the presence of 2 mm MgCl2. After the addition of 500 μl of TBS (Tris-buffered saline; 50 mm Tris buffer, pH 7.6, 150 mm NaCl), the VLPs were centrifuged at 16,000 × g for 1 h at 4 °C, resuspended in 50 μl of RNase-free water and 3 μl of 20 mg/ml proteinase K (Invitrogen), and incubated at 55 °C for 30 min. Next, 200 μl of 5.8 m guanidinium isothiocyanate and 10 μl of 20 mg/ml glycogen were added and incubated for 5 min at room temperature. RNA samples were precipitated with isopropyl alcohol, centrifuged for 30 min at 21,000 × g, and the RNA pellets were dissolved in RNase-free water. Quantification of total RNA from VLPs was performed using the Ribogreen Quantification Kit (Molecular Probes, Inc.). Quantification of HIV-1 RNA in VLPs was performed using reverse transcriptase-real time PCR as described previously (28Gorelick R.J. Gagliardi T.D. Bosche W.J. Wiltrout T.A. Coren L.V. Chabot D.J. Lifson J.D. Henderson L.E. Arthur L.O. Virology. 1999; 256: 92-104Crossref PubMed Scopus (122) Google Scholar). Briefly, cDNA synthesis was performed using random primers, and real time PCR was carried out in the presence of the U5-Ψ primer-probe set of HIV-1 (29Svarovskaia E.S. Barr R. Zhang X. Pais G.C. Marchand C. Pommier Y. Burke Jr., T.R. Pathak V.K. J. Virol. 2004; 78: 3210-3222Crossref PubMed Scopus (96) Google Scholar). Immunofluorescence Staining of Transfected Cells—293T cells were cultured on poly-l-lysine-coated 12-mm-diameter coverslips (BD Biosciences) for 24 h prior to transfection. At 8 h post-transfection, cells were fixed for 20 min at room temperature in 5% paraformadehyde and 2% sucrose in PBS and permeabilized with 1% Triton X-100 and 10% sucrose in PBS for 30 min. We used anti-c-Myc monoclonal antibody clone 9E10 (Sigma) as primary antibody and Alexa Fluor 568-conjugated goat antibody to mouse IgG(H+L) (Molecular Probes) as secondary fluorescent antibody. Cells were incubated sequentially with each antibody for 1 h and washed three times. All washes and antibody dilutions were done in PBS containing 1% bovine serum albumin (Sigma), 1% Triton X-100 (Sigma), and 2% normal goat serum (Vector Laboratories, Inc.). Finally, the nuclei were stained by incubating the cells in PBS containing 2 μg/ml 4′,6-diamidino-2-phenyl-indole (DAPI) (Sigma) for 2 min followed by two washes in PBS. The coverslips were rinsed in deionized water and then mounted on glass slides by using FluoroGuard (Bio-Rad). Cells were visualized using a LSM510 Zeiss confocal fluorescence microscope (Carl Zeiss). Coimmunoprecipitation Analysis—An anti-c-Myc antibody (Sigma) was coupled to paramagnetic beads according to the manufacturer's instructions (Dynal Biotech). 293T cells were cotransfected with APOBEC3G and pGag; pGag expressed HIV-1 Gag that possessed a FLAG tag epitope at its C terminus. Approximately 36 h after transfection, 4 × 106 cells were harvested, washed twice with ice-cold PBS, and lysed in 2 ml of cell extraction buffer (20 mm Tris-Cl, pH 8.0, 137 mm NaCl, 1 mm EDTA, 1 mm NaVO3, 10% glycerol, 1% Triton X-100, and protease inhibitor mixture (Roche Applied Science)). The cell extracts were centrifuged at 1,500 × g for 4 min, and the supernatant was divided equally into two samples; 100 μg of RNase A (Sigma) was added to one sample, and 100 units of RNaseOUT (Invitrogen) was added to the other sample. The samples were incubated for 20 min at room temperature and subsequently incubated with anti-c-Myc antibody-conjugated paramagnetic beads for 3 h in slow rotation on RKDynal rotor (Dynal Biotech) at 4 °C. After incubation, the paramagnetic beads were washed three times with 50 mm Tris-HCl, pH 7.5, 500 mm LiCl, 1 mm NaVO3, and 0.5% Triton X-100, three times with 50 mm Tris-HCl, pH 7.5, 500 mm LiCl, 1 mm NaVO3, and once with 1 mm NaVO3. The bound proteins were eluted from the beads by heating to 90 °C for 5 min in SDS-PAGE loading buffer and analyzed by Western blotting for the presence of APOBEC3G using the anti-c-Myc antibody and Gag using an anti-FLAG antibody (Sigma). Western Blotting Analysis—293T cells were transfected with 20 μgof pHDV-EGFP, 8 μg of pcDNA-APO3G, and 8 μg of pcDNA3.1-eIF3k-c-Myc (kindly provided by John W. B. Hershey) (31Mayeur G.L. Fraser C.S. Peiretti F. Block K.L. Hershey J.W. Eur. J. Biochem. 2003; 270: 4133-4139Crossref PubMed Scopus (48) Google Scholar). The virus-producing cells and the VLPs were harvested 36 h later, lysed in 1 × SDS-PAGE loading buffer, denatured at 96 °C for 10 min, and analyzed using anti-c-Myc antibody (Sigma) by Western blotting as described previously (13Xu 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). To gain insights into the mechanism by which APOBEC3G is incorporated into retroviral particles, we developed a sensitive and quantitative cytidine deamination assay using scintillation proximity beads (Fig. 1A). Briefly, an oligonucleotide primer containing a 5′-biotinylated deoxynucleotide is hybridized to a template oligonucleotide containing deoxycytidines and deoxyadenines (e.g. (CCCA)10) and attached to scintillation proximity beads. The template-primer complex is incubated with HIV-1 VLPs containing APOBEC3G for 1–6 h at 37 °C. Next, the primer is extended using Klenow fragment in the presence of either a [3H]dATP, dGTP, and dTTP mixture or a [3H]dTTP, dGTP, and dATP mixture. [3H]dATP is incorporated into the nascent DNA only upon conversion of deoxycytidines to deoxyuridines, providing a measure of the cytidine deamination activity. To control for efficiency of Klenow fragment-mediated DNA synthesis, incorporation of [3H]dTTP is also determined. Incorporation of [3H]dATP or [3H]dTTP in the extended primer brings the radiolabeled nucleotide in close proximity to the scintillation bead, resulting in a signal that can easily be quantified. An additional positive control reaction is performed in parallel using a (UUUA)10 template to determine the maximal [3H]dATP incorporation level, which was set to 100% and referred to as APO activity. The APO activity for experimental samples is expressed as a percentage of the [3H]dATP incorporation relative to the (UUUA)10 template. To determine whether APOBEC3G and cytidine deamination activity is present in cell culture supernatants in the absence of HIV-1 proteins, we transfected 293T cells with pcDNA-APO3G plasmid and determined whether the culture supernatants contained cytidine deamination activity (Fig. 1B). A very low level of cytidine deamination activity (<2%) was detected, indicating that APOBEC3G release from 293T cells required HIV-1 proteins. To determine whether other cytidine deaminases expressed in 293T cells contributed to the cytidine deamination activity, we transfected 293T cells with an HIV-1 gag-pol expression construct, isolated VLPs from culture supernatants, and determined cytidine deamination activity. In the absence of transfected APOBEC3G plasmid, very low levels of cytidine deamination activity were detected (<1%), indicating that cytidine deaminases expressed from 293T cells did not contribute significantly to the observed cytidine deamination activity. To determine which template sequences provided an optimal substrate for cytidine deamination, we compared the cytidine deamination activity after transfections with an HIV-1 gag-pol expression construct and an APOBEC3G expression plasmid on (CCA)13, (CCCA)10, and (CCCCA)8 templates (Fig. 1B). The results indicated that the (CCCA)10 template was the most efficient substrate for detection of cytidine deaminase activity; therefore, this template was used in all subsequent experiments. Next, we determined how the quantity of VLPs and time of incubation of template with APOBEC3G correlated with the observed cytidine deamination activity (Fig. 1C). VLPs produced from cells transfected with HIV-1 gag-pol and APOBEC3G expression constructs were quantified by determining the amount of p24 CA levels by enzyme-linked immunosorbent assay; 0–45 ng of p24 CA associated with VLPs was incubated with the (CCCA)10 template-primer complex for 1, 2, and 6 h, and the extent of cytidine deamination was determined. The results indicated that the cytidine deamination activity increased as the amounts of CA associated with VLPs or time of incubation increased and leveled off at either high VLP input or longer incubation times (e.g. 12 ng of p24 CA for 6 h or 24 ng of p24 CA for 2 h). The data indicated that ∼6 ng of p24 CA associated with VLPs for 2 h resulted in ∼30% APO activity, which was within the linear range of detection. Therefore, APOBEC3G activities were determined for ≤6 ng of p24 CA associated with VLPs for 2 h in subsequent experiments. To determine whether APOBEC3G is packaged into HIV-1 VLPs specifically or nonspecifically, we compared the intracellular distribution and relative efficiencies of virion incorporation of APOBEC3G and cytoplasmic translational initiation factor 3 subunit k (eIF3k) (31Mayeur G.L. Fraser C.S. Peiretti F. Block K.L. Hershey J.W. Eur. J. Biochem. 2003; 270: 4133-4139Crossref PubMed Scopus (48) Google Scholar). To compare the intracellular distributions of APOBEC3G and eIF3k, the plasmids that express each protein were transfected into 293T cells; the transfected cells were stained with an anti-c-Myc tag antibody and analyzed by confocal microscopy (Fig. 2A). The results indicated that both APOBEC3G and eIF3k appeared to be distributed throughout the cytoplasm of the transfected cells. To compa

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