(A) TZM-bl indicator cells were infected with WT and Gag30 mutant SIVcpz and HIV-1 constructs as indicated. Infections were performed in triplicate with virus stocks containing 1 ng of p24 antigen (average values ± SDs are shown). CPZa, chimpanzee adapted. (B) Relative infectivity of WT and Gag30 mutant HIV-1 and SIVcpz constructs. Shown are minimum and maximum values, 25% and 75% percentiles, and median values. (C) Modulation of viral infectivity by host-specific adaptation at Gag30. Changing a Lys or Arg to a Met or Leu reduced the infectivity of HIV-1 by 50%, while the reciprocal mutations more than doubled the infectivity of SIVcpz. Values are shown in relation to those of the corresponding parental HIV-1 or SIVcpz constructs set to 100%.

(A) Representative growth curves of WT and Gag30 mutant HIV-1, CPZ-adapted HIV-1 (CPZa), and SIVcpz constructs. Infections were performed in triplicate. Data shown are average values ± SD. (B) Virus production by tonsillary explant cultures infected with SIVcpz and HIV-1 constructs. Cumulative virus production is shown over 14 days. Matched tissues from 7 to 12 donors were inoculated with the WT HIV-1 (left), chimpanzee-adapted HIV-1 (middle). and SIVcpz (right) constructs or with their respective Gag30 mutants, and for each HLT culture, virus production was measured over 14 days. Data are shown as mean ± SEM. (C) Average cumulative p24 antigen levels in HLT cultures from 7 to 12 donors. Data are shown as mean ± SEM. (D) Host-specific adaptation increases replication fitness. Mutation of K/R30M/L reduces HIV-1 replication, and substitution of M/L30R increases SIVcpz replication in HLTs. Cumulative p24 antigen production levels of the Gag30 mutants are shown in relation to those of their respective parental constructs set to 100%.

The 4 molecular clones of SIVcpz analyzed in this study are highlighted in yellow. The tree was constructed using maximum likelihood methods *Bootstrap support of greater than 80%. Scale bar: 0.1 amino acid replacements per site.

(A) Replication kinetics are shown for isogenic pairs of WT and Gag30 mutant HIV-1 (red) and SIVcpz (green) clones in human (HU) and chimpanzee (CPZ) CD4⁺ T lymphocytes. Each virus pair was tested in cells from 6 chimpanzee and 4 human donors by measuring RT activity in culture supernatants (one representative curve is shown). (B) Competition of WT and Gag30 mutant HIV-1 and SIVcpz constructs. Human CD4⁺ T cells were infected with virus stocks that were normalized for p24 content and mixed at a 1:1 ratio. Sequence chromatograms of RT-PCR amplification products obtained from the input virus and cell culture supernatants collected at 7 days after infection are shown. (C) Cumulative virus production over 13 days of culture in CD4⁺ T lymphocytes from 4 human (left) and 6 chimpanzee (right) donors infected with the indicated HIV-1 or SIVcpz variants. Data are shown as mean ± SEM. (D) Mean production of HIV-1 or SIVcpz constructs containing an R/K or M/L at Gag30 in human- or chimpanzee-derived CD4⁺ T cells. Data are shown as mean ± SEM obtained for all 3 HIV-1 and 4 SIVcpz strains analyzed. (E) Replication of SIVcpz M/L30R mutants in human and chimpanzee cells. Cumulative RT production levels of the Gag30 mutants are shown in relation to those of their parental constructs set to 100%. WT JRCSF and YU2C strains did not replicate at detectable levels in most chimpanzee cells, thus precluding a direct comparison.

(A) Detection of WT and mutant sequences in viral mixtures. Left: WT and K30M mutant HIV-1 NL4-3 stocks were normalized for p24 content, mixed at different ratios as indicated, and sequenced directly. Right: example for a standard curve. The peak fluorescence of the 2 WT A residues is expressed as a fraction of the total fluorescence. (B) Sequence chromatograms of mixed viral cultures at input and 14 days after infection. Percentages of WT and mutant sequences provided in B were estimated from standard curves. Values of less than 10% may represent nonspecific background.