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A Single Amino Acid in Human APOBEC3F Alters Susceptibility to HIV-1 Vif*

  • John S. Albin
    Footnotes
    Affiliations
    Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455

    Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota 55455

    Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455
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  • Rebecca S. LaRue
    Footnotes
    Affiliations
    Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455

    Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota 55455

    Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455
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  • Jessalyn A. Weaver
    Affiliations
    Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455

    Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota 55455

    Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455
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  • William L. Brown
    Affiliations
    Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455

    Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota 55455

    Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455
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  • Keisuke Shindo
    Affiliations
    Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455

    Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota 55455

    Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455
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  • Elena Harjes
    Affiliations
    Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455

    Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota 55455
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  • Hiroshi Matsuo
    Affiliations
    Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455

    Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota 55455
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  • Reuben S. Harris
    Correspondence
    To whom correspondence should be addressed: 6-155 Jackson Hall, 321 Church St. S.E., Minneapolis, MN 55455. Tel.: 612-624-0457; Fax: 612-625-2163
    Affiliations
    Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455

    Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota 55455

    Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455
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  • Author Footnotes
    * This work was supported, in whole or in part, by the National Institutes of Health Grant R01 AI064046 through the NIAID (to R. S. H.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.
    1 Supported by the National Institute on Drug Abuse (F30 DA026310) and the University of Minnesota Medical Scientist Training Program (T32 GM008244).
    2 Supported in part by a studentship from the Minnesota Agricultural Experiment Station and the College of Veterinary Medicine.
      Human APOBEC3F (huA3F) potently restricts the infectivity of HIV-1 in the absence of the viral accessory protein virion infectivity factor (Vif). Vif functions to preserve viral infectivity by triggering the degradation of huA3F but not rhesus macaque A3F (rhA3F). Here, we use a combination of deletions, chimeras, and systematic mutagenesis between huA3F and rhA3F to identify Glu324 as a critical determinant of huA3F susceptibility to HIV-1 Vif-mediated degradation. A structural model of the C-terminal deaminase domain of huA3F indicates that Glu324 is a surface residue within the α4 helix adjacent to residues corresponding to other known Vif susceptibility determinants in APOBEC3G and APOBEC3H. This structural clustering suggests that Vif may bind a conserved surface present in multiple APOBEC3 proteins.

      Introduction

      Human APOBEC3 proteins including APOBEC3F (huA3F) and APOBEC3G (huA3G) are DNA cytidine deaminases that restrict the infectivity of HIV-1 in target cells following virion incorporation in producer cells (recently reviewed in Refs.
      • Romani B.
      • Engelbrecht S.
      • Glashoff R.H.
      ,
      • Henriet S.
      • Mercenne G.
      • Bernacchi S.
      • Paillart J.C.
      • Marquet R.
      ,
      • Albin J.S.
      • Harris R.S.
      ). HIV-1 overcomes this restriction activity by utilizing its accessory protein virion infectivity factor (Vif)
      The abbreviations used are: Vif
      virion infectivity factor
      agm
      African green monkey
      CTD
      C-terminal deaminase domain
      hu
      human
      NTD
      N-terminal deaminase domain
      rh
      rhesus
      SIV
      simian immunodeficiency virus.
      to facilitate the degradation of APOBEC3 proteins in producer cells, thus preventing particle incorporation and restriction.
      Previously, several groups identified specific changes in the N-terminal deaminase domain (NTD) of huA3G that affect the ability of HIV-1 Vif to neutralize this restriction factor (
      • Mangeat B.
      • Turelli P.
      • Liao S.
      • Trono D.
      ,
      • Bogerd H.P.
      • Doehle B.P.
      • Wiegand H.L.
      • Cullen B.R.
      ,
      • Schröfelbauer B.
      • Chen D.
      • Landau N.R.
      ,
      • Xu H.
      • Svarovskaia E.S.
      • Barr R.
      • Zhang Y.
      • Khan M.A.
      • Strebel K.
      • Pathak V.K.
      ,
      • Huthoff H.
      • Malim M.H.
      ,
      • Russell R.A.
      • Smith J.
      • Barr R.
      • Bhattacharyya D.
      • Pathak V.K.
      ,
      • Lavens D.
      • Peelman F.
      • Van der Heyden J.
      • Uyttendaele I.
      • Catteeuw D.
      • Verhee A.
      • Van Schoubroeck B.
      • Kurth J.
      • Hallenberger S.
      • Clayton R.
      • Tavernier J.
      ). The first of these studies sought to determine the basis for the observation that the Vif proteins of the lentiviruses infecting different species neutralize the A3G proteins of their natural host species but not the A3G proteins of other species (
      • Mariani R.
      • Chen D.
      • Schröfelbauer B.
      • Navarro F.
      • König R.
      • Bollman B.
      • Münk C.
      • Nymark-McMahon H.
      • Landau N.R.
      ). For example, African green monkey A3G (agmA3G) is susceptible to Vif from the simian immunodeficiency virus (SIV) that naturally infects Chlorocebus aethiops (agmSIV) but not to HIV-1 Vif, whereas huA3G is susceptible to HIV-1 Vif but not to agmSIV Vif. By substituting agmA3G residues into huA3G where the two differed, several groups identified Asp128 as a critical determinant of this species specificity (
      • Mangeat B.
      • Turelli P.
      • Liao S.
      • Trono D.
      ,
      • Bogerd H.P.
      • Doehle B.P.
      • Wiegand H.L.
      • Cullen B.R.
      ,
      • Schröfelbauer B.
      • Chen D.
      • Landau N.R.
      ,
      • Xu H.
      • Svarovskaia E.S.
      • Barr R.
      • Zhang Y.
      • Khan M.A.
      • Strebel K.
      • Pathak V.K.
      ). Subsequent mutational analyses have confirmed that huA3G Asp128 and surrounding residues including Asp130 impact HIV-1 Vif-mediated degradation (
      • Huthoff H.
      • Malim M.H.
      ,
      • Russell R.A.
      • Smith J.
      • Barr R.
      • Bhattacharyya D.
      • Pathak V.K.
      ,
      • Lavens D.
      • Peelman F.
      • Van der Heyden J.
      • Uyttendaele I.
      • Catteeuw D.
      • Verhee A.
      • Van Schoubroeck B.
      • Kurth J.
      • Hallenberger S.
      • Clayton R.
      • Tavernier J.
      ).
      More recently, two reports showed that, in contrast with huA3G, huA3F is recognized at its C-terminal deaminase domain (CTD) by HIV-1 Vif (
      • Russell R.A.
      • Smith J.
      • Barr R.
      • Bhattacharyya D.
      • Pathak V.K.
      ,
      • Zhang W.
      • Chen G.
      • Niewiadomska A.M.
      • Xu R.
      • Yu X.F.
      ). One of these groups further narrowed the determinants of this recognition to amino acids 283–300, although individual amino acid changes critical for HIV-1 Vif susceptibility were not identified in a manner analogous to the huA3G studies cited above. Thus, the residues of huA3F critical for the ability of HIV-1 Vif to bind and degrade this restriction factor are presently unknown.
      Here, we identify a critical determinant of huA3F susceptibility to HIV-1 Vif by comparing huA3F with the closely related but HIV-1 Vif-resistant rhA3F (
      • Virgen C.A.
      • Hatziioannou T.
      ,
      • Zennou V.
      • Bieniasz P.D.
      ). Using chimeras between these orthologs as well as single-domain studies, we confirm that Vif recognizes the CTD of huA3F. Through systematic replacement of selected C-terminal huA3F residues with their corresponding rhA3F residues, we further identify huA3F Gln323/Glu324 as a critical determinant of this differential susceptibility. Additional mutagenesis between these two residues revealed that mutation of Glu324 to the rhA3F lysine or to alanine results in resistance to HIV-1 Vif-mediated degradation. To determine the three-dimensional context surrounding this residue, we created a model of the CTD of huA3F and found that Glu324 is a surface residue contained within the α4 helix that forms part of a broader surface shared with the linearly separate huA3F Vif interaction domain previously narrowed to residues 283–300 (
      • Russell R.A.
      • Smith J.
      • Barr R.
      • Bhattacharyya D.
      • Pathak V.K.
      ). Importantly, this analysis also revealed that the huA3F residues corresponding to three known Vif susceptibility determinants, Asp128 and Asp130 in huA3G and Asp/Glu121 in human APOBEC3H (huA3H), also cluster at this helix. These studies combine to suggest that a conserved structural surface is targeted by HIV-1 Vif en route to APOBEC3 neutralization and degradation.

      EXPERIMENTAL PROCEDURES

       Plasmid DNA Construction and Site-directed Mutagenesis

      All constructs were confirmed by DNA sequencing. huA3F and huA3G coding sequences correspond to those found in GenBank NM_145298 and NM_021822, respectively. rhA3F was provided by Dr. Theodora Hatziioannou (Aaron Diamond AIDS Research Center, New York) (
      • Virgen C.A.
      • Hatziioannou T.
      ,
      • Zennou V.
      • Bieniasz P.D.
      ). Substitutions of rhA3F residues into huA3F were based on alignment between huA3F and rhA3F reference sequences NM_145298 and NM_001042373.1. pcDNA3.1-V5, -huA3F-V5, and -huA3G-V5 have been described, and pcDNA3.1-huA3G-V5 D128K was similarly derived (
      • Albin J.S.
      • Haché G.
      • Hultquist J.F.
      • Brown W.L.
      • Harris R.S.
      ).
      A3F domain chimeras were made using overlapping PCR (
      • LaRue R.S.
      • Jónsson S.R.
      • Silverstein K.A.
      • Lajoie M.
      • Bertrand D.
      • El-Mabrouk N.
      • Hötzel I.
      • Andrésdóttir V.
      • Smith T.P.
      • Harris R.S.
      ). PCR products were digested with KpnI/XhoI and ligated into similarly cut pcDNA3.1-V5.
      Single domains of huA3F and rhA3F were amplified using primers containing SacI/SalI sites and cloned into similarly cut pEGFP-N3 (Clontech). Full-length huA3F-GFP has been described (
      • Stenglein M.D.
      • Harris R.S.
      ); NTD = residues 1–191; CTD = residues 192–373.
      huA3F P281L/E282D, N289K/T300A, T303A, and D313H were introduced into the pcDNA3.1-huA3F-3×HA construct (
      • Stenglein M.D.
      • Harris R.S.
      ) by site-directed mutagenesis using Pfu polymerases (Stratagene). The 3×HA tag was subsequently replaced with a V5 tag (
      • Albin J.S.
      • Haché G.
      • Hultquist J.F.
      • Brown W.L.
      • Harris R.S.
      ). All other mutations were introduced directly into pcDNA3.1-huA3F-V5.
      HIV-1IIIB and SIVmac239 Vif as well as a vector derived from pVR1012 have been described (
      • Albin J.S.
      • Haché G.
      • Hultquist J.F.
      • Brown W.L.
      • Harris R.S.
      ,
      • LaRue R.S.
      • Lengyel J.A.
      • Jónsson S.R.
      • Andrésdóttir V.
      • Harris R.S.
      ). An untagged, codon-optimized version of HIV-1IIIB Vif was made by PCR amplification and ligation of the coding region into the SalI/BamHI segment of the original construct. The codon-optimized translated Vif open reading frames are those of HIV-1IIIB (GenBank EU541617) and SIVmac239 (GenBank AY588946).
      Proviral plasmid HIV-1IIIB is a nucleotide A200C derivative of pIIIB (
      • Albin J.S.
      • Haché G.
      • Hultquist J.F.
      • Brown W.L.
      • Harris R.S.
      ). A Vif-deficient A200C pIIIB derivative containing a previously described deletion in vif made by overlap extension PCR was used in spreading infections (
      • Gibbs J.S.
      • Regier D.A.
      • Desrosiers R.C.
      ). A Vif-deficient pIIIB derivative containing tandem stop codons at positions 26–27 of vif was used for all single-cycle infectivity experiments and has been described previously (
      • Albin J.S.
      • Haché G.
      • Hultquist J.F.
      • Brown W.L.
      • Harris R.S.
      ,
      • Haché G.
      • Shindo K.
      • Albin J.S.
      • Harris R.S.
      ). Wild-type and Vif-deficient LAI-GFP were kindly provided by Dr. Mario Stevenson (University of Massachusetts, Worcester, MA).

       Cell Lines

      293T cells were maintained in DMEM supplemented with 10% fetal bovine serum and, in some cases, penicillin/streptomycin. CEM-GFP reporter cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin/streptomycin, and β-mercaptoethanol (
      • Haché G.
      • Shindo K.
      • Albin J.S.
      • Harris R.S.
      ,
      • Gervaix A.
      • West D.
      • Leoni L.M.
      • Richman D.D.
      • Wong-Staal F.
      • Corbeil J.
      ).

       Stability of A3F Chimeras in the Presence of HIV-1 and SIV Vifs

      At 50% confluence in 6-well plates, 293T cells were transfected using Trans-IT transfection reagent (Mirus Bio) with 100 ng of A3-V5 and 25 ng of Vif-HA. After 48 h, cell lysates were harvested and resuspended in 2 × sample buffer (25 mm Tris, pH 6.8, 8% glycerol, 0.8% SDS, 2% β-mercaptoethanol, 0.02% bromphenol blue), boiled for 10 min, and run on a 12% SDS-polyacrylamide gel prior to transfer to a PVDF membrane (Millipore). Membranes were probed with mouse anti-V5 (Invitrogen), mouse anti-HA.11 (Covance), or mouse anti-α-tubulin (Covance) primary antibodies followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies. Membranes were developed using HyGLO chemiluminescent HRP detection reagent (Denville Scientific) and exposed to film. Blots were stripped using 0.2 m glycine, 1.0% SDS, 1.0% Tween 20, pH 2.2, between sequential probing with primary antibodies.

       Single-cycle Infectivity Assays

      250,000 293T cells were plated in 2 ml of DMEM in 6-well plates. One day later, Trans-IT transfection reagent was used to cotransfect these cells with 1.6 μg Vif-deficient HIV-1IIIB, 100 ng of a codon-optimized HIV-1IIIB Vif-HA expression construct (or 50–200 ng supplemented to 200 ng total with a vector control in Fig. 2C), and 200 ng of a given APOBEC3-V5 construct. Approximately 2 days later, virus-containing supernatants were filtered through 0.45-μm PVDF filters (Millipore), and 75 or 150 μl was used to infect 25,000 CEM-GFP reporter cells plated at a final total volume of 250 μl. At this time, 293T cells were resuspended with 1 ml of phosphate-buffered saline (PBS), and 500 μl was spun down and resuspended in 250 μl of lysis buffer (25 mm HEPES, pH 7.4, 150 mm NaCl, 1 mm MgCl2, 50 μm ZnCl2, 10% glycerol, and 1% Triton X-100 supplemented with 50 μm MG132 (American Peptide) and complete protease inhibitor (Roche Applied Science)) for analysis of APOBEC3 intracellular stability. Three days after harvest, CEM-GFP reporter cells were fixed with 4% paraformaldehyde and analyzed by flow cytometry on a Beckman-Coulter Quanta MPL or a Becton-Dickinson LSR II to determine infectivity as measured by the percentage of GFP-positive cells. Relative infectivity was calculated by normalizing the infectivity of viruses produced in the presence of each APOBEC3 protein ± Vif to the infectivity of viruses produced in the presence of an APOBEC3 vector control ± Vif in each experiment. Data shown represent the mean ± S.E. of the number of independent transfection-infection series indicated in the figure legends.
      Figure thumbnail gr2
      FIGURE 2Substitution of rhA3F residues at positions 323–324 of huA3F results in phenotypic Vif resistance. A, schematic depiction of the CTDs of huA3F and rhA3F with relevant amino acids shown. B, single-cycle infectivity data quantifying the effects of HIV-1 Vif on the rescue of HIV-1 infectivity. All mutants remained competent for restriction. Relative infectivity represents the mean ± S.E. of four independent experiments. Western blots demonstrating intracellular APOBEC3 and Vif levels corresponding to one of these experiments are shown below. C, single-cycle infectivity data demonstrating the continued restriction of HIV-1 by untagged huA3F Q323E/E324K and huA3G D128K/D130K in the presence of increasing amounts of Vif. Relative infectivity represents the mean ± S.E. (error bars) of two independent experiments done in duplicate. The Western blots demonstrating the stability of Vif-resistant huA3F and huA3G variants in the presence of Vif correspond to one of these infectivity experiments. D, Western blots showing the expression levels of huA3F, huA3G, and their Vif-resistant variants in the SupT11 cell lines used in E. E, representative spreading infection curves demonstrating that wild-type HIV-1IIIB is restricted by Vif-resistant variants huA3F Q323E/E324K and huA3G D128K/D130K but not the corresponding wild-type APOBEC3 proteins. Open symbols indicate vector control or huA3F- or huA3G-expressing cell lines. Filled symbols indicate cell lines expressing huA3F Q323E/E324K or huA3G D128K/D130K. The x axis is offset from zero to permit visualization of curves yielding no detectable spread.

       Analysis of Intracellular APOBEC3 Stability

      Cotransfected cells were lysed as described above, and aliquots were mixed with a 5 × or 7.5 × version of the sample buffer described to a final concentration of 2 × and boiled for 10 min. Proteins were then separated by 10% SDS-PAGE and transferred to a PVDF membrane. Membranes were probed as described above and stripped using 62.5 mm Tris, pH 6.8, 2% SDS, and 100 mm β-mercaptoethanol at 50 °C prior to sequential blocking and reprobing.

       Analysis of Intracellular APOBEC3 Expression

      APOBEC3-expressing and vector control derivatives of SupT11 as well as the model nonpermissive cell lines CEM and H9 were grown to confluence in 10-cm dishes. 5 × 106 cells were then lysed in 250 μl and analyzed for huA3F or huA3G expression using antibodies 1474 or 10201 from Drs. Michael Malim or Jaisri Lingappa, respectively, obtained through the AIDS Research and Reference Reagent Program.

       Virus Titration and Spreading Infections

      Viruses were produced by plating 3.5 × 106 293T cells in 10-cm dishes and 1 day later transfecting those cells with 5–10 μg of a given proviral plasmid using Trans-IT transfection reagent. Approximately 2 days after transfection, supernatants were harvested, and different volumes were used to infect 150,000 CEM-GFP reporter cells at a constant total volume of 1 ml in 24-well plates. Three days later, these CEM-GFP cells were fixed in 4% paraformaldehyde, and the total percentage of GFP-positive cells was quantified by flow cytometry as before. Linear regression was then employed to determine the volume of a given viral stock required to initiate infection at a CEM-GFP multiplicity of infection of 0.01.
      Spreading infections were initiated by infecting 150,000 cells of a given cell line at a multiplicity of infection of 0.01 in a total volume of 1 ml in 24-well plates. Cultures were subsequently split and fed as necessary to prevent cell overgrowth. Viral spread was monitored by periodically harvesting 150 μl of supernatant from each culture and using it to infect 25,000 CEM-GFP cells at a final volume of 250 μl in 96-well plates. Three days after infection, these CEM-GFP were fixed in 4% paraformaldehyde, and the percentage of GFP-positive cells was analyzed by flow cytometry on a Becton-Dickinson LSR II.

       Homology Modeling of the huA3F C-terminal Deaminase Domain

      The A3F186–373 model was generated using YASARA (
      • Krieger E.
      • Koraimann G.
      • Vriend G.
      ) based on the crystal structure of A3G191–384 2K3A (Protein Data Bank code 3IR2) (
      • Shandilya S.M.
      • Nalam M.N.
      • Nalivaika E.A.
      • Gross P.J.
      • Valesano J.C.
      • Shindo K.
      • Li M.
      • Munson M.
      • Royer W.E.
      • Harjes E.
      • Kono T.
      • Matsuo H.
      • Harris R.S.
      • Somasundaran M.
      • Schiffer C.A.
      ). Alignment with the A3G191–384 sequence (supplemental Fig. 1) was iteratively optimized using related SwissProt and TrEMBL sequences, the predicted secondary structure and the structural information of the template. Knowledge-based and electrostatic interactions in unrestrained molecular dynamics with explicit solvent molecules were used to refine amino acid side chain geometry. Insertions were accounted for by a search of the Protein Data Bank for loop ends superimposable with model anchor points. Further optimization was achieved by placement of loops into their lowest energy conformations.

       Coimmunoprecipitation

      Coimmunoprecipitation of V5-tagged huA3F with HA-tagged HIV-1 Vif or with HA-tagged Vif with mutation of the conserved BC Box residues SLQ>AAA that ablate Vif-mediated degradation was carried out by lysing cotransfected 293T cells with radioimmune precipitation assay buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS supplemented with complete protease inhibitor (Roche Applied Science)). Lysates were then incubated with 2.5 μl of mouse anti-HA.11 at 4 °C followed by the addition of 40 μl of Dynabeads protein G (Invitrogen). Immunoprecipitated complexes were isolated by magnetic separation, washed four times with PBS, and eluted by addition of 30 μl of 5 × sample buffer as above. SDS-PAGE and Western blotting were then carried out as before. Western blots were quantified by analysis with ImageJ software (National Institutes of Health). Binding quantification represents the intensity of V5 bands immunoprecipitated divided by the intensity of the corresponding lysate bands and normalized to the ratio found for huA3F in a given experiment.

      RESULTS

       HIV-1 Vif Recognizes the huA3F C-terminal Deaminase Domain

      Two previous reports have indicated that HIV-1 Vif binds the CTD of huA3F (
      • Russell R.A.
      • Smith J.
      • Barr R.
      • Bhattacharyya D.
      • Pathak V.K.
      ,
      • Zhang W.
      • Chen G.
      • Niewiadomska A.M.
      • Xu R.
      • Yu X.F.
      ). To confirm and extend these results, we took two approaches. First, we created chimeras between huA3F and rhA3F, which share 87% identity and 92% overall similarity at the protein level (Fig. 1A). This strategy takes advantage of the fact that rhA3F is resistant to HIV-1 Vif whereas huA3F is sensitive to HIV-1 Vif (
      • Virgen C.A.
      • Hatziioannou T.
      ,
      • Zennou V.
      • Bieniasz P.D.
      ). Therefore, by comparing the sensitivity of chimeric proteins to HIV-1 Vif-mediated degradation, one can broadly infer whether a given chimera contains a site functionally recognized by HIV-1 Vif. As shown in Fig. 1B, huA3F and the rhA3F/huA3F chimera containing the rhA3F NTD and the huA3F CTD retained high sensitivity to HIV-1IIIB Vif-mediated degradation on cotransfection of a given chimera with HIV-1 Vif. In contrast, rhA3F and the huA3F/rhA3F chimera containing the huA3F NTD and the rhA3F CTD were insensitive to the presence of HIV-1 Vif. To confirm these results, we cotransfected HA-tagged HIV-1 Vif or SIV Vif with GFP-tagged single deaminase domains of huA3F or rhA3F, respectively, and assessed the stability of each domain. Both HIV-1 Vif and SIV Vif destabilized the CTDs of huA3F and rhA3F, respectively, whereas the corresponding NTDs were relatively unaffected (Fig. 1, C and D). Thus, our data corroborate prior reports demonstrating that the A3F CTD is necessary and sufficient for Vif-mediated degradation (
      • Russell R.A.
      • Smith J.
      • Barr R.
      • Bhattacharyya D.
      • Pathak V.K.
      ,
      • Zhang W.
      • Chen G.
      • Niewiadomska A.M.
      • Xu R.
      • Yu X.F.
      ).
      Figure thumbnail gr1
      FIGURE 1Susceptibility of huA3F to HIV-1 Vif maps to the huA3F CTD. A, schematic depiction of the chimeras used in B. B, cotransfection experiment demonstrating the instability in the presence of HIV-1 Vif of chimeras between rhA3F and huA3F that contain the huA3F CTD. C and D, cotransfection experiments demonstrating that the CTDs of huA3F and rhA3F are destabilized by HIV-1 Vif or SIV Vif, respectively, whereas the corresponding NTDs remains highly expressed.

       A3F Residues 323–324 Affect the Susceptibility of huA3F to HIV-1 Vif

      To map more closely the residues critical for functional neutralization of huA3F, we created a series of huA3F mutants containing 1 or 2 rhA3F residues at sites where these two differ within their CTDs. A schematic of the substitutions made is shown in Fig. 2A. To test the Vif susceptibility of these mutants, we carried out single-cycle infectivity assays. As shown in Fig. 2B, infectivity restoration upon the cotransfection of HIV-1IIIB Vif with most huA3F mutants was similar to that seen with wild-type huA3F. For the substitution Q323E/E324K, however, Vif sensitivity was ablated (Fig. 2B). These infectivity data correlated with producer cell huA3F levels, making this mutant phenotypically analogous to the control huA3G D128K.
      Interestingly, neither of these substitutions is contained within the Vif binding region proposed by Russell et al. (
      • Russell R.A.
      • Smith J.
      • Barr R.
      • Bhattacharyya D.
      • Pathak V.K.
      ), residues 283–300, whereas the substitutions that do fall within this region (N298K/T298A) have no apparent phenotype (Fig. 2, A and B, and “Discussion”). Furthermore, huA3F D313H, which corresponds to the change D130K in the evolutionarily related NTD of huA3G, has no apparent phenotype (Fig. 2, A and B, and “Discussion”) (
      • LaRue R.S.
      • Andrésdóttir V.
      • Blanchard Y.
      • Conticello S.G.
      • Derse D.
      • Emerman M.
      • Greene W.C.
      • Jónsson S.R.
      • Landau N.R.
      • Löchelt M.
      • Malik H.S.
      • Malim M.H.
      • Münk C.
      • O'Brien S.J.
      • Pathak V.K.
      • Strebel K.
      • Wain-Hobson S.
      • Yu X.F.
      • Yuhki N.
      • Harris R.S.
      ).
      To confirm the intrinsic Vif resistance of huA3F Q323E/E324K and eliminate the possibility that the C-terminal V5 tag initially used might affect our observations, we carried out single-cycle titration experiments using increasing levels of Vif cotransfected with a constant amount of untagged huA3F, huA3F Q323E/E324K, huA3G or huA3G D128K/D130K. As shown in Fig. 2C, both huA3F Q323E/E324K and huA3G D128K/D130K retained similar restriction regardless of Vif levels. The Vif resistance of both constructs was further confirmed by the intracellular stability of each in comparison with its wild-type control in the presence of Vif.
      Because the single-cycle infectivity assays described to this point are vulnerable to potential overexpression artifacts (e.g.
      • Browne E.P.
      • Allers C.
      • Landau N.R.
      ,
      • Miyagi E.
      • Opi S.
      • Takeuchi H.
      • Khan M.
      • Goila-Gaur R.
      • Kao S.
      • Strebel K.
      ,
      • Schumacher A.J.
      • Haché G.
      • MacDuff D.A.
      • Brown W.L.
      • Harris R.S.
      ), we also sought to assess the Vif resistance of the huA3F Q323E/E324K construct in a more physiologic setting. To that end, we created derivatives of a previously described APOBEC3-deficient T cell line, SupT11, stably transfected with untagged huA3F Q323E/E324K or huA3G D128K/D130K to go with our previously described derivatives expressing wild-type huA3F or huA3G (
      • Albin J.S.
      • Haché G.
      • Hultquist J.F.
      • Brown W.L.
      • Harris R.S.
      ,
      • Refsland E.W.
      • Stenglein M.D.
      • Shindo K.
      • Albin J.S.
      • Brown W.L.
      • Harris R.S.
      ). The expression levels of huA3F and huA3G in each cell line used are shown in Fig. 2D. We then initiated spreading infections at a multiplicity of infection of 0.01 on these cell lines using Vif-deficient or wild-type IIIB or LAI-GFP viruses. All cell lines with the exception of the vector controls restricted the spread of Vif-deficient HIV-1IIIB and HIV-1LAI-GFP (data not shown). In contrast, both wild-type HIV-1IIIB and HIV-1LAI-GFP, which differ at 20/192 Vif amino acids, spread efficiently on cell lines expressing wild-type huA3F or huA3G (Fig. 2E and data not shown). Despite this, similar levels of huA3F Q323E/E324K or huA3G D128K/D130K restricted the spread of even these Vif-proficient viruses (Fig. 2E and data not shown). We thus conclude that huA3F Q323E/E324K, like huA3G D128K/D130K, is fully resistant to HIV-1 Vif and fully capable of inhibiting virus replication.

       Reciprocal Amino Acid Substitutions rhA3F E323Q/K324E Do Not Sensitize rhA3F to HIV-1 Vif

      To determine whether the reciprocal amino acid substitutions in rhA3F might render it susceptible to HIV-1 Vif in a manner analogous to the sensitization of agmA3G by the humanizing mutation K128D (
      • Mangeat B.
      • Turelli P.
      • Liao S.
      • Trono D.
      ,
      • Bogerd H.P.
      • Doehle B.P.
      • Wiegand H.L.
      • Cullen B.R.
      ,
      • Schröfelbauer B.
      • Chen D.
      • Landau N.R.
      ,
      • Xu H.
      • Svarovskaia E.S.
      • Barr R.
      • Zhang Y.
      • Khan M.A.
      • Strebel K.
      • Pathak V.K.
      ), we carried out single-cycle infectivity and expression analyses as above using rhA3F and rhA3F E323Q/K324E. Under these conditions, rhA3F E323Q/K324E showed no significant recovery in infectivity in the presence of HIV-1 Vif over wild-type rhA3F (Fig. 3). Thus, residues 323–324 are not exclusively responsible for the differential Vif sensitivity of rhA3F and huA3F. This observation is consistent with the emerging view that a larger surface on APOBEC3 proteins is recognized by Vif (see “Discussion”).
      Figure thumbnail gr3
      FIGURE 3Substitution of human residues at positions 323–324 of rhA3F does not sensitize rhA3F to HIV-1 Vif. Single-cycle infectivity experiments demonstrate that substituting the human residues at positions 323–324 of rhA3F does not sensitize this restriction factor to permit infectivity recovery in the presence of Vif. Data represent the mean and S.E. (error bars) of three independent experiments. Western blots corresponding to one of the single-cycle experiments shown demonstrate the correlation between intracellular stability of APOBEC3 variants and functional recovery in infectivity.
      It is important to note that such separation of function experiments are not possible using SIVmac239 Vif, because this Vif neutralizes both huA3F and rhA3F (
      • Zennou V.
      • Bieniasz P.D.
      ). This parallels the results of several of the original papers characterizing huA3G D128K, which show that SIVmac239 Vif is able to neutralize both huA3G and rhA3G (e.g.
      • Schröfelbauer B.
      • Chen D.
      • Landau N.R.
      ,
      • Xu H.
      • Svarovskaia E.S.
      • Barr R.
      • Zhang Y.
      • Khan M.A.
      • Strebel K.
      • Pathak V.K.
      ). It is also consistent with our own studies suggesting that the Vifs of various species' lentiviruses are optimized for recognition of their own host species' APOBEC3Z3 proteins but often retain considerable activity against the APOBEC3Z3 proteins of other species (
      • LaRue R.S.
      • Lengyel J.A.
      • Jónsson S.R.
      • Andrésdóttir V.
      • Harris R.S.
      ).

       Mutation of huA3F Glu324 Alone Alters Functional Susceptibility to HIV-1 Vif in the Absence of a Quantitative Reduction in Physical Binding

      To characterize further the changes at residues 323–324, we created huA3F mutants with single cognate rhesus substitutions at each position as well as single and double alanine mutations at these positions and assessed their restriction activities and Vif susceptibilities as before. This analysis revealed that any huA3F variant lacking glutamate at position 324 is resistant to HIV-1 Vif regardless of the identity of residue 323, which correlates with intracellular A3F levels (Fig. 4A). We therefore conclude that residue 324 is a single amino acid determinant of huA3F HIV-1 Vif susceptibility.
      Figure thumbnail gr4
      FIGURE 4The identity of residue 324 is a primary determinant of the degradation sensitivity of huA3F to HIV-1 Vif. A, single-cycle infectivity data quantifying the restriction and Vif-sensitivity phenotypes of single and double human-to-rhesus and human-to-alanine mutations at positions 323 and/or 324 of huA3F, where only mutations at position 324 ablate Vif responsiveness. Data represent the mean ± S.E. (error bars) of three independent experiments. Western blots corresponding to one of these experiments demonstrating that mutations at position 324 of huA3F result in resistance to Vif-mediated degradation are shown below. B, representative experiment demonstrating the lack of effect of mutations at huA3F positions 323–324 on coimmunoprecipitation with HA-tagged Vif. The bands in each row are taken unaltered from different parts of the same blot. C, quantification of the results from a total of five independent experiments including the one shown in B. Relative binding represents the ratio of IP V5 signal to cellular V5 signal normalized to the ratio observed for A3F in a given experiment (set to 1).
      To determine whether mutation of Glu324 alters the quantitative binding of Vif to huA3F, we cotransfected HA-tagged Vif with V5-tagged huA3F and variants mutated at residues 323 and 324. We then immunoprecipitated HA-tagged Vif from these lysates and blotted for associated V5-tagged huA3F. Despite the resistance of huA3F Glu324 variants to HIV-1 Vif-mediated degradation, however, we found that mutation of this residue does not reduce coimmunoprecipitation with HIV-1 Vif relative to wild type (Fig. 4, B and C). These results were confirmed by reciprocal immunoprecipitation of V5-tagged huA3F or huA3F E324K and blotting for cotransfected, untagged Vif in both RIPA and Nonidet P-40 lysis buffers (data not shown).

       Determinants of HIV-1 Vif Recognition Localize to the α4 Helix of a Susceptible Deaminase Domain

      To visualize Glu324 in its three-dimensional context, we created a model of the CTD of huA3F based on a recent crystal structure of the huA3G CTD (Protein Data Bank code 3IR2) (
      • Shandilya S.M.
      • Nalam M.N.
      • Nalivaika E.A.
      • Gross P.J.
      • Valesano J.C.
      • Shindo K.
      • Li M.
      • Munson M.
      • Royer W.E.
      • Harjes E.
      • Kono T.
      • Matsuo H.
      • Harris R.S.
      • Somasundaran M.
      • Schiffer C.A.
      ). As shown in Fig. 5, A and B, Glu324 is located on the surface of the α4 helix. Importantly, this region is also adjacent to the linearly separate stretch of amino acids previously implicated in the huA3F interaction with HIV-1 Vif, residues 283–300 (
      • Russell R.A.
      • Smith J.
      • Barr R.
      • Bhattacharyya D.
      • Pathak V.K.
      ). We also noted, however, that several additional negatively charged residues occurred on or near the surface of this helix: Asp311, Asp313, and Glu316. On aligning these residues to those found in other Vif-susceptible deaminase domains in Fig. 5C, we noted that they each align to a previously described APOBEC3 determinant of Vif susceptibility (
      • Mangeat B.
      • Turelli P.
      • Liao S.
      • Trono D.
      ,
      • Bogerd H.P.
      • Doehle B.P.
      • Wiegand H.L.
      • Cullen B.R.
      ,
      • Schröfelbauer B.
      • Chen D.
      • Landau N.R.
      ,
      • Xu H.
      • Svarovskaia E.S.
      • Barr R.
      • Zhang Y.
      • Khan M.A.
      • Strebel K.
      • Pathak V.K.
      ,
      • Huthoff H.
      • Malim M.H.
      ,
      • Russell R.A.
      • Smith J.
      • Barr R.
      • Bhattacharyya D.
      • Pathak V.K.
      ,
      • Lavens D.
      • Peelman F.
      • Van der Heyden J.
      • Uyttendaele I.
      • Catteeuw D.
      • Verhee A.
      • Van Schoubroeck B.
      • Kurth J.
      • Hallenberger S.
      • Clayton R.
      • Tavernier J.
      ,
      • Zhen A.
      • Wang T.
      • Zhao K.
      • Xiong Y.
      • Yu X.F.
      ). Asp311 and Asp313 in huA3F correspond to Asp128 and Asp130 in the NTD of huA3G, whereas huA3F E316 corresponds to Asp/Glu121 in human APOBEC3H (huA3H). Thus, all currently known APOBEC3 determinants of Vif susceptibility cluster along the surface of the α4 helix, and all are negatively charged.
      Figure thumbnail gr5
      FIGURE 5Model structure of the CTD of huA3F. A, ribbon diagram depicting the CTD of huA3F. The region encompassing the huA3F equivalents of all known single amino acid determinants of Vif sensitivity is shown in blue, with Asp311, Asp313, and Glu316 shown in orange and Glu324 in red. The region previously implicated in huA3F interaction with Vif (residues 283–300) is colored purple. B, predicted surface of the huA3F CTD. C, alignment of residues in the α4 helix encompassing known determinants of Vif susceptibility.

      DISCUSSION

      The studies described here are the first to identify a single amino acid determinant of the susceptibility of huA3F to HIV-1 Vif. This represents an important advance in our understanding of the HIV-1 Vif-huA3F interaction, the relevance of which is strongly supported by a large body of work demonstrating the potency of huA3F-mediated restriction of HIV-1 (e.g.
      • Bishop K.N.
      • Holmes R.K.
      • Sheehy A.M.
      • Davidson N.O.
      • Cho S.J.
      • Malim M.H.
      ,
      • Liddament M.T.
      • Brown W.L.
      • Schumacher A.J.
      • Harris R.S.
      ,
      • Wiegand H.L.
      • Doehle B.P.
      • Bogerd H.P.
      • Cullen B.R.
      ,
      • Zheng Y.H.
      • Irwin D.
      • Kurosu T.
      • Tokunaga K.
      • Sata T.
      • Peterlin B.M.
      ). Our own long term viral evolution studies have also suggested that functional neutralization of huA3F by HIV-1 Vif is required for the virus propagate in the presence of huA3F (
      • Albin J.S.
      • Haché G.
      • Hultquist J.F.
      • Brown W.L.
      • Harris R.S.
      ). Thus, shielding the α4 region of huA3F described here from HIV-1 Vif may represent a viable strategy for the development of novel pharmacotherapies for HIV-1 infection (
      • Albin J.S.
      • Harris R.S.
      ,
      • Harris R.S.
      • Liddament M.T.
      ).
      Our work confirms prior reports that broadly localized Vif interaction to the CTD of huA3F (
      • Russell R.A.
      • Smith J.
      • Barr R.
      • Bhattacharyya D.
      • Pathak V.K.
      ,
      • Zhang W.
      • Chen G.
      • Niewiadomska A.M.
      • Xu R.
      • Yu X.F.
      ) (Fig. 1). An additional recent report on the existence of a Vif-susceptible splice variant of huA3F composed largely of the CTD is also consistent with these data (
      • Lassen K.G.
      • Wissing S.
      • Lobritz M.A.
      • Santiago M.
      • Greene W.C.
      ).
      Our identification of a single amino acid determinant of HIV-1 Vif susceptibility in huA3F echoes several prior reports localizing Vif susceptibility in huA3G and huA3H in that the residue identified is a single negative charge localized to the surface of an APOBEC3 protein (
      • Mangeat B.
      • Turelli P.
      • Liao S.
      • Trono D.
      ,
      • Bogerd H.P.
      • Doehle B.P.
      • Wiegand H.L.
      • Cullen B.R.
      ,
      • Schröfelbauer B.
      • Chen D.
      • Landau N.R.
      ,
      • Xu H.
      • Svarovskaia E.S.
      • Barr R.
      • Zhang Y.
      • Khan M.A.
      • Strebel K.
      • Pathak V.K.
      ) (FIGURE 4, FIGURE 5). Glu324 differs from these reports in one key respect, however, as a charge substitution was involved in all prior reports. For example, huA3G D128A has no phenotype, whereas D128K is Vif-resistant (e.g.
      • Bogerd H.P.
      • Doehle B.P.
      • Wiegand H.L.
      • Cullen B.R.
      ,
      • Schröfelbauer B.
      • Chen D.
      • Landau N.R.
      ,
      • Huthoff H.
      • Malim M.H.
      ). The fact that both alanine and lysine substitutions at huA3F Glu324 ablate Vif susceptibility (Fig. 4A) suggests that Glu324 is required either for overall stability of the broader Vif binding surface or for direct functional interaction with HIV-1 Vif. Changes to this residue do not, however, affect the ability of huA3F to restrict HIV-1; in fact, none of the changes described in these studies affected restriction activity (e.g. FIGURE 2, FIGURE 3, FIGURE 4).
      It is notable that Glu324 does not fall within the region previously found by Russell et al. to be critical for HIV-1 Vif recognition of huA3F (
      • Russell R.A.
      • Smith J.
      • Barr R.
      • Bhattacharyya D.
      • Pathak V.K.
      ). These residues, 283–300, encompass most of the α3 helix, which is structurally adjacent to α4 and Glu324 and appears to form a common surface (Fig. 5, A and B) (
      • Russell R.A.
      • Smith J.
      • Barr R.
      • Bhattacharyya D.
      • Pathak V.K.
      ). It is therefore possible that Glu324 may cooperate with residues in α3 and/or α4 to create a stable surface recognized by HIV-1 Vif, in which case mutational alteration of any critical component of this putative Vif interaction node may affect Vif sensitivity. Alternatively, Russell et al. used chimeras between huA3F and huA3G to map the huA3F Vif-interacting region (
      • Russell R.A.
      • Smith J.
      • Barr R.
      • Bhattacharyya D.
      • Pathak V.K.
      ). The CTDs of huA3F and huA3G, however, are evolutionarily divergent Z2 and Z1 types, respectively (
      • LaRue R.S.
      • Andrésdóttir V.
      • Blanchard Y.
      • Conticello S.G.
      • Derse D.
      • Emerman M.
      • Greene W.C.
      • Jónsson S.R.
      • Landau N.R.
      • Löchelt M.
      • Malik H.S.
      • Malim M.H.
      • Münk C.
      • O'Brien S.J.
      • Pathak V.K.
      • Strebel K.
      • Wain-Hobson S.
      • Yu X.F.
      • Yuhki N.
      • Harris R.S.
      ). This means that chimeras in this domain will contain a relatively large number of amino acid substitutions versus wild type. For example, only half of residues 283–300 are biochemically similar or identical between huA3F and huA3G. Thus, we think it likely that the structure of huA3F/huA3G chimeras in this region will be altered relative to huA3F, which may affect interaction with Vif.
      In addition to suggesting the structural unity of our findings with those of Russell et al., our model of the huA3F CTD allowed us to make an important observation about the nature of the region surrounding Glu324; namely, both the α4 and the neighboring α3 helices have a number of negatively charged surface residues (Fig. 5, A and B). This led us to align these surface residues with those in other Vif-susceptible APOBEC3 deaminase domains such as the huA3G NTD and huA3H, which showed that each negatively charged surface residue in the α4 helix of huA3F corresponds to a known negatively charged determinant of Vif susceptibility in another APOBEC3 protein (Fig. 5). Thus, although determining the identity of all the amino acid residues with which Vif interacts (i.e. the broader Vif binding surface in APOBEC3 proteins) will require a great deal of future genetic and structural study, it is intriguing that all known Vif susceptibility determinants map to the same structural motif. This implies a degree of structural conservation among Vif-APOBEC3 interaction surfaces that would not be apparent from a simple linear comparison of these single amino acid determinants.
      Although the lack of functional interaction between Vif and huA3F Glu324 variants is clear, this appears to be due to a qualitative change in the nature of the Vif-huA3F interaction in Glu324 mutants because coimmunoprecipitation of huA3F Glu324 mutants with HIV-1 Vif is unimpaired. Our data therefore support the potential for both qualitative and quantitative changes in huA3F binding to Vif that may alter susceptibility. In the absence of structural data, however, we are unable to explain the nature of the qualitative defect found in huA3F Glu324 mutants. It is possible that the qualitative defect in Glu324 mutants may involve a conformational change that prevents productive interaction with HIV-1 Vif. Alternatively, mutation of Glu324 may impair the recruitment of other components of the E3 ligase complex by HIV-1 Vif en route to degradation. It is also conceivable that Glu324 mutants may have a functionally relevant, altered affinity for HIV-1 Vif that is not readily apparent by coimmunoprecipitation.
      In summary, we have described here a single amino acid determinant of huA3F susceptibility to HIV-1 Vif. This advance in our understanding of the Vif-huA3F interaction echoes the single amino acid determinants previously identified in other APOBEC3 proteins, as all are negatively charged residues that may interact directly with the highly basic Vif protein. Importantly, the observation that all of these single amino acid determinants cluster along the α4 helix raises the exciting possibility that certain features of the Vif-APOBEC3 interaction may be structurally conserved, which would facilitate the design of hypothetical single molecules which may simultaneously block the functional interaction of Vif with multiple APOBEC3 proteins. Indeed, although its exact mechanism of action remains unknown, the compound RN-18 provides proof of concept for just such a scenario (
      • Nathans R.
      • Cao H.
      • Sharova N.
      • Ali A.
      • Sharkey M.
      • Stranska R.
      • Stevenson M.
      • Rana T.M.
      ).

      Acknowledgments

      We thank T. Hatziioannou, P. Bieniasz, X.F. Yu and the AIDS Research and Reference Reagent Program for materials, D. Urso for technical assistance, L. Lackey for helpful discussion and L. Mansky for sharing flow cytometry facilities.

      Supplementary Material

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