Expression of exogenous APOBEC3G-HA is sufficient to render CEM-T4 cells non-permissive for Vif-deficient HIV-1.

<p>(A) An immunoblot showing APOBEC3G-HA expression in CEM-T4 clones G8, G9, and G11 but not in vector control clones V1, V8, and V12. These clones were generated by electroporating an APOBEC3G-HA or vector-control plasmid into CEM-T4-A cells, followed by serial dilution in 96-well plates and outgrowth in selective media containing G418. The anti-HA and anti-tubulin (TUB) antibodies were purchased from Covance. (B and C) Replication kinetics of Vif-deficient or Vif-proficient HIV-1, respectively, on the indicated clones. Solid lines and filled symbols represent virus replication on APOBEC3G-expressing clones and dashed lines and open symbols represent virus replication on vector-control clones.</p

CEM-T4 cells express low APOBEC3G levels and subclones show additional heterogeneity.

<p>(A) An immunoblot showing APOBEC3G expression levels in CEM, CEM-SS, CEM-T4, and CEM-T4 subclones. The CEM-T4 line was obtained from both the AIDS Research and Reference Reagent Program (courtesy of Dr. J. P. Jacobs) and Y.-H. Zheng (Michigan State University <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000528#ppat.1000528-Han1" target="_blank">[4]</a>), designated CEM-T4-A and CEM-T4-Z, respectively. CEM-T4 subclones A1-5 and Z1-5 were obtained by expanding single cells. The anti-APOBEC3G (A3G) antibody was provided by J. Lingappa (University of Washington) through the AIDS Research and Reference Reagent Program, and the anti-tubulin (TUB) antibody was from Covance. (B and C) Replication kinetics of Vif-deficient HIV-1 (IIIB <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000528#ppat.1000528-Hach1" target="_blank">[8]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000528#ppat.1000528-Sheehy1" target="_blank">[9]</a>) on the indicated lines or subclones. Virus replication was monitored by mixing cell-free culture supernatant with the reporter cells CEM-GFP, and infectivity was determined by determining the percentage of GFP-positive CEM-GFP cells by flow cytometry <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000528#ppat.1000528-Gervaix1" target="_blank">[7]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000528#ppat.1000528-Hach1" target="_blank">[8]</a>.</p

An evolutionary wobble model accounts for the dynamic nature of the interactions between the lentivirus Vif protein and host APOBEC3 enzymes.

<p>(<b>A</b>) Space-filled representation of the Vif/CBF-β complex (PDB 4N9F). Yellow, orange, and red shading highlights Vif-binding surfaces unique to A3D/F, A3G, and A3H, respectively. The A3D and A3F interaction surfaces are largely overlapping due to high levels of homology caused by a recent gene duplication. (<b>B</b>) A schematic of the “Wobble Model” for adaptation of lentiviral Vif. Left to right: The ancestral Vif-APOBEC3 interaction is predicted to be strong (illustrated by six interaction hexagons). Virus transmission to a new host with a larger APOBEC3 repertoire requires a major adaptive change, such as the binding of CBF-β during the inferred ancient lentivirus transmission from an ancestral cat to a prosimian. This event most likely reshaped Vif and attenuated the Vif-APOBEC3 interaction. However, it provided physical substrate for a series of rapid adaptations to independently strengthen each Vif-APOBEC3 interaction. The present-day, largely non-overlapping surfaces of HIV-1 Vif that interact with A3D/F, A3G, and A3H are attributable to slower and continuous adaptations driven by adaptive immune pressures.</p

Lentiviral Vif and APOBEC3 diversity and functionality in mammals.

<p>(<b>A</b>) The primate lentiviral Vif/CBF-β complex protects viral genetic integrity by polyubiquitinating and degrading cellular APOBEC3 enzymes. (<b>B</b>) Structural representations of the Vif/CBF-β and RUNX/CBF-β heterodimeric complexes (PDB 4N9F and 1H9D, respectively). CBF-β is positioned similarly in each schematic to highlight the extensive shared interaction surface. (<b>C</b>) A schematic depicting the relative phylogenetic relationships between present-day lentivirus-infected mammals and APOBEC3 domain copy numbers (clockwise from top left, humans, non-human primates, cats, cattle, goats, and sheep; simple tree generated by Phylot). The subset of APOBEC3 domains known to interact with each species’ Vif is shaded orange. Primate lentiviruses use Vif proteins that require CBF-β, whereas non-primate lentiviruses do not (these viruses appear to lack an analogous cofactor [FIV/BIV] or to have adapted to exploit a different cellular cofactor [CypA for MVV/CAEV] [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005958#ppat.1005958.ref026" target="_blank">26</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005958#ppat.1005958.ref027" target="_blank">27</a>]). One-way arrows from cats to non-human primates and from non-human primates to humans depict cross-species lentivirus transmission events. (<b>D</b>) An illustration depicting the dual-functionality of primate lentiviral Vif (APOBEC3 protein degradation and <i>APOBEC3</i> gene expression inhibition) versus the mono-functionality of non-primate lentiviral Vif (APOBEC3 protein degradation).</p

Gene targeting statistics in CEM2n.

<p>Gene targeting statistics in CEM2n.</p

Characterization of independent knockout and knockdown clones.

<p>(A) Levels of each indicated <i>A3</i> mRNA in CEM2n, <i>A3G</i>- and <i>A3F</i>-null derivatives, and CEM-SS (relative to <i>TBP</i>; mean and s.d. shown for triplicate experiments). (B) Levels of each indicated <i>A3</i> mRNA in CEM2n or <i>A3F</i>-null cells transduced with shNS, shA3D, or shA3G constructs (relative to <i>TBP</i>; mean and s.d. shown for triplicate experiments). (C) Single-cycle infectivity of Vif-deficient HIV produced in parallel in the indicated cell lines (mean and s.d. shown for p24-normalized triplicate experiments). (D) The kinetics of Vif-proficient (blue diamonds) and Vif-deficient (red squares) HIV spreading infection in the indicated cell lines. Numbers distinguish independent clones.</p

Construction and characterization of <i>A3F</i>-Null CEM2n cells.

<p>(A) <i>A3F</i> exon 3–4 targeting strategy. LA, left homology arm; SA, splice acceptor; IRES, internal ribosomal entry site; Neo, G418 resistance gene; pA, poly adenylation signal; RA, right homology arm; yellow triangles, <i>loxP</i> sites. (B) <i>A3</i> mRNA expression profiles of the indicated cells relative to parental CEM2n (mean and s.d. shown for triplicate experiments). (C) Immunoblots of A3F, A3G, and tubulin (TUB) in the indicated cells. (D) Infectivity of Vif-deficient HIV produced using the indicated cell lines following a single replicative cycle (mean and s.d. shown for p24-normalized triplicate experiments). (E) 3D-PCR profiles of HIV <i>gag-pol</i> and cellular <i>MDM2</i> targets within genomic DNA of infected CEM-GFP reporter cells. (F) HIV G-to-A mutation profiles of proviruses originating in the indicated cell types. The mutation frequency at each dinucleotide is illustrated as a pie chart wedge (n≥15 kb per condition).</p

Mutation summary.

<p>Mutation summary.</p

Construction and characterization of <i>A3F</i>-Null/<i>A3</i>-Knockdown CEM2n cells.

<p>(A) Levels of each indicated <i>A3</i> mRNA in CEM2n or <i>A3F</i>-null cells transduced with shNS, shA3B, shA3C, shA3D, shA3G, or shA3H constructs (mean and s.d. shown for triplicate experiments). (B) Immunoblots of A3G and tubulin (TUB) in CEM2n or <i>A3F</i>-null cells stably transduced with the indicated shRNA-expressing lentivirus. (C) Infectivity of Vif-deficient HIV produced using the indicated transduced cell pool and reported using the CEM-GFP system (mean and s.d. shown for p24-normalized triplicate experiments; in some instances, the error is nearly indistinguishable from the histogram bar outline). (D) 3D-PCR profiles of HIV <i>gag-pol</i> and cellular <i>MDM2</i> targets within genomic DNA of infected CEM-GFP reporter cells. (E) HIV G-to-A mutation profiles of proviruses originating in the indicated cell types. The mutation frequency at each dinucleotide is illustrated as a pie chart wedge (n≥15 kb per condition). Pie charts were generated for those conditions with ≥1 mutation per kb analyzed. Mutation numbers for all conditions can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002800#ppat-1002800-t002" target="_blank">Table 2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002800#ppat.1002800.s007" target="_blank">Table S1</a>.</p

Construction and characterization of <i>A3G</i>-Null CEM2n cells.

<p>(A) <i>A3G</i> exon 3 targeting strategy. LA, left homology arm; SA, splice acceptor; IRES, internal ribosomal entry site; Neo, G418 resistance gene; pA, poly adenylation signal; RA, right homology arm; yellow triangles, <i>loxP</i> sites. (B) <i>A3</i> mRNA expression profiles of the indicated cells relative to parental CEM2n (mean and s.d. shown for triplicate experiments). (C) Immunoblots of A3G, A3F, and tubulin (TUB) in the indicated cells. (D) Infectivity of Vif-deficient HIV produced using the indicated cell lines following a single replicative cycle (mean and s.d. shown for p24-normalized triplicate experiments). (E) 3D-PCR profiles of HIV <i>gag-pol</i> and cellular <i>MDM2</i> targets within genomic DNA of infected CEM-GFP reporter cells. (F) HIV G-to-A mutation profiles of proviruses originating in the indicated cell types. The mutation frequency at each dinucleotide is illustrated as a pie chart wedge (n≥15 kb per condition).</p