Fig 2.

Identification of amino acid residues in HIV-1mt CA that are critical for viral growth enhancement in macaque cells. (A) Alignment of Gag-CA sequences. CA amino acid sequences of HIV-1mt MN4Rh-3 (19, 24), HIV-2 GL-AN (72), and SIVmac239 MA239 (71) are aligned. The N-terminal β-hairpin and helices 1 to 11 are indicated based on previously reported analyses (50, 73). The CA-Q110D mutation in MN4Rh-3 (19, 24) is shaded. Substitutions of three amino acids that contribute to the enhancement of HIV-1mt growth in macaque cells, described in this work, are indicated by arrows. (B) Growth kinetics of a parental clone, MN4Rh-3, and its CA mutants carrying a single-amino-acid change (see Table 1 for the mutants). Viruses were prepared from 293T cells transfected with the proviral clones indicated, and equal amounts (2 × 10⁶ RT units) were inoculated into M1.3S cells (3 × 10⁵ cells). Virus replication was monitored by RT activity released into the culture supernatants. Representative data from two independent experiments are shown. (C) Schematic CA structure of HIV-1mt clones. Amino acid substitutions are indicated in the order in which they were introduced into HIV-1mt CA. Black areas show sequences from SIVmac239. Helices 4 to 7 are shown as gray areas with the helix number. (D) Growth kinetics of CXCR4-tropic (left) and CCR5-tropic (right) HIV-1mt clones with different CA proteins (see reference 19 for CCR5-tropic MN5Rh-3). Viruses were prepared from 293T cells transfected with the indicated proviral clones, and equal amounts (2.5 × 10⁶ RT units) were inoculated into M1.3S cells (10⁶ cells). Virus replication was monitored by RT activity released into the culture supernatants. Representative data from two independent experiments are shown.