Similar anti-SIVcpz activity of cpzA3H haplotypes.

<p>(<b>a</b>) Four single nucleotide polymorphisms (SNPs) of cpzA3H were observed in 61 chimpanzees, see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006746#ppat.1006746.t001" target="_blank">Table 1</a>. These four SNPs including reference cpzA3H were named haplotype I (hapI) to haplotype V (hapV). CDD, cytidine deaminase domain. (<b>b</b>) The polygenetic relationship of primate A3H proteins. 500 bootstrap replications were performed during calculation. The number on each node indicates the bootstrap support. (<b>c</b>) cpzA3H expression plasmids for different haplotypes were transfected into 293T cells. After two days, the expression of cpzA3H was detected by using anti-HA antibody, respectively. Tubulin served as a loading control. (<b>d</b>) SIVcpz<i>Ptt</i>MB897WT or SIVcpz<i>Ptt</i>MB897Δ<i>vif</i> reporter constructs were co-transfected with expression plasmids for cpzA3H haplotypes into 293T cells, pcDNA3.1(+) empty vector was used as control (vector). Two days post-transfection, normalized amounts of virus were used to infect 293T cells. The nanoluciferase (relative light units-RLU) was measured 2 days post-infection. (<b>e</b>) The expression plasmids of cpzA3H haplotypes were co-transfected with expression plasmids for Vifs (from different SIVcpz strains, as indicated) into 293T cells. The presence of A3H and Vif were detected by using anti-HA and anti-Vif antibodies, respectively. Tubulin served as a loading control.</p

Stable hA3H inhibits SIVcpz replication in human PBMCs.

<p>(<b>a</b>) The genotypes of different donors were determined by sequencing the A3H mRNAs. Expression of A3H and A3G proteins in stimulated PBMCs were detected by immunoblots using anti-hA3H and anti-hA3G antibodies, respectively. Tubulin served as a loading control. (<b>b, c</b>) PBMCs from different donors were infected with 1 ng RT or 5 ng RT activity of SIVcpz<i>Ptt</i>MB897, respectively, and culture supernatants were collected each 2–3 day and analyzed for the RT activity.</p

Residue 97 of A3H partially determines the sensitivity to SIVcpz Vifs.

<p>(<b>a, b</b>) SIVcpz<i>Ptt</i>MB897 or SIVcpz<i>Pts</i>TAN1 wild type or delta v<i>if</i> reporter viruses were produced in 293T cells in the presence of A3H wild type or mutant expression plasmids, as indicated. The viral infectivity was determined by measuring the nanoluciferase of 293T cells infected with normalized amounts of SIVcpz reporter viruses. (<b>c, d</b>) cpzA3H, hA3H wild type or mutants expression plasmids were co-transfected with SIVcpz (SIVcpz<i>Ptt</i>MB897 or SIVcpz<i>Pts</i>TAN1) Vif expression plasmids into 293T cells. The presence of A3H and Vif was detected by using anti-HA and anti-Vif antibodies, respectively. Tubulin served as a loading control.</p

Human APOBEC3s (hA3s) inhibit SIVs.

<p>(<b>a, b</b>) SIVmac or SIVagm wild type or delta <i>vif</i> reporter viruses were produced in 293T cells in the presence of hA3s. PTR600 empty vector was used as control (vector) for hA3s. Two days post-transfection, normalized amounts of viruses were used to infect 293T cells, firefly luciferase (relative light units-RLU) was measured two days post-infection. (<b>c, d</b>) SIVcpz<i>Ptt</i>MB897 or SIVcpz<i>Pts</i>TAN1 wild type or delta <i>vif</i> reporter viruses were produced in 293T cells in the presence of hA3s. PTR600 empty vector was used as control (vector) for hA3s. Two days post-transfection, normalized amounts of viruses were used to infect 293T cells. Two days post-infection, 293T cells were carefully washed once with PBS, and nanoluciferase (relative light units-RLU) was measured. The relative infectivity was shown. Values are means plus standard deviations (error bars) of a representative experiment performed in triplicate. Asterisks represent statistically significant differences: P value < 0.001 extremely significant (***), 0.001 to 0.01 very significant (**), 0.01 to 0.05 significant (*), >0.05 not significant (ns).</p

Chimpanzee APOBEC3s (cpzA3s) inhibit SIVs.

<p>(<b>a, b</b>) SIVmac or SIVagm wild type or delta <i>vif</i> reporter viruses were produced in 293T cells in the presence of cpzA3s. pcDNA3.1(+) was used as control (vector) for cpzA3s. Two days post-transfection, normalized amounts of viruses were used to infect 293T cells, firefly luciferase (relative light units-RLU) was measured two days post-infection. (<b>c, d</b>) SIVcpz<i>Ptt</i>MB897 or SIVcpz<i>Pts</i>TAN1 wild type or delta <i>vif</i> reporter viruses were produced in 293T cells in the presence of cpzA3s. pcDNA3.1(+) was used as control (vector) for cpzA3s. Two days post-transfection, normalized amounts of viruses were used to infect 293T cells. Two days post-infection, 293T cells were carefully washed once with PBS, and nanoluciferase (relative light units-RLU) was measured, relative infectivity was shown. Values are means plus standard deviations (error bars) of a representative experiment performed in triplicate. Asterisks represent statistically significant differences: P value < 0.001 extremely significant (***), 0.001 to 0.01 very significant (**), 0.01 to 0.05 significant (*), >0.05 not significant (ns).</p

The model of SIVcpz cross species transmission to human.

<p>The model predicts that cross-species transmission of SIVcpz is blocked by hA3H hapII (or other stably expressed variants), but this transmission is easier obtained in humans with unstable A3H haplotypes.</p

Single round infection assay of SIVcpz reporter viruses.

<p>SIVcpz-NLuc was produced in 293T cells in the presence (+) or absence (-) of VSV-G expression plasmid. Virions that had 20 pg reverse transcription activity were used for infection of 293T cells, viral infectivity was determined by quantification of nanoluciferase activity in lysates of infected cells, cps counts per second.</p

Genetic variants in the APOBEC3H gene in chimpanzee subspecies.

<p>Genetic variants in the APOBEC3H gene in chimpanzee subspecies.</p

Inhibition of SIVcpz by hA3H haplotypes with stable protein expression.

<p>(<b>a</b>) SIVcpz<i>Ptt</i>MB897WT or SIVcpz<i>Ptt</i>MB897Δ<i>vif</i> reporter constructs were co-transfected with increasing amounts of hA3H hapI or hapII expression plasmids (5, 10, 30, 100 or 200 ng), PTR600 empty vector was used to bring the total transfected plasmid DNA to 600 ng and also used as control (vector). Two days post-transfection, normalized amounts of viruses were used to infect 293T cells. The nanoluciferase (relative light units-RLU) was measured two days post-infection. (<b>b</b>) Cell lysates from (a) were used to detect the expression of A3s, SIVcpz capsid (p24), or SIVcpz Vif by anti-HA, anti-p24, or anti-Vif antibody, respectively. Tubulin served as a loading control. (<b>c</b>) SIVcpz<i>Ptt</i>MB897WT or SIVcpz<i>Ptt</i>MB897Δ<i>vif</i> reporter constructs were co-transfected but with expression plasmids for different A3H haplotypes and splice variants. (<b>d</b>) Cell lysates from (c) were used to detect the expression of A3s by anti-HA antibody. Tubulin served as a loading control. (<b>e</b>) SupT11-vetor-hCCR5 or SupT11-hA3H hapII-hCCR5 cells were infected with 1 ng RT activity of SIVcpz<i>Pts</i>TAN1, SIVcpz<i>Ptt</i>MB897 or SIVcpz<i>Ptt</i>Gab1, respectively, and culture supernatants were collected each second day and quantified by the RT assay.</p

No counteraction of human A3H haplotype II by Vifs from different SIVcpz lineages.

<p>(<b>a</b>) cpzA3H or hA3H hapII wild type expression plasmids were co-transfected with expression plasmids for Vifs (different SIVcpz, SIVgor or HIV-1 strains, as indicated). The presence of A3H and Vif was detected by using anti-HA and anti-Vif antibodies, respectively. Tubulin served as a loading control. (<b>b</b>) The schematic structure of SIVcpz<i>Ptt</i>MB897 and HIV-1 LAI Vif chimeras. (<b>c</b>) hA3H hapII and Vif chimera expression plasmids were co-transfected into 293T cells. The presence of A3H and Vif was detected by using anti-HA and anti-Vif antibodies, respectively. Tubulin served as a loading control. (<b>d</b>) Schematic structure of SIVcpz<i>Ptt</i>MB897 Vif mutants. (<b>e</b>) cpzA3H or hA3H hapII wild type expression plasmids were co-transfected with expression plasmids for different SIVcpz<i>Ptt</i>MB897 Vif mutants. The presence of A3H and Vif was detected by using anti-HA and anti-Vif antibodies, respectively. Tubulin served as a loading control. (<b>f</b>) SIVcpz<i>Ptt</i>MB897 with the mutation of 47EN48-47PH48 in Vif and wild type SIVcpz (5 ng RT activity) were used to infect SupT11-vetor-hCCR5 or SupT11-hA3H hapII-hCCR5 cells, respectively. The viral supernatants were collected each second day and quantified by RT assay. (<b>g</b>) Superimposition of SIVcpz Vif-hA3H hapII model structure. 47EN48 and 60GDAK63 motifs are shown by sphere, and their contact with hA3H hapII is displayed. (<b>h</b>) Summary: Correlation between Vif from different SIVcpz and HIV-1 lineages and antagonism activity against cpzA3H and hA3H hapII. Identity of amino acids at important positions (39, 47–48 and 60–63) of Vif was shown. -, + represents low and high A3H antagonism.</p