A single amino acid of the human immunodeficiency virus type 2 capsid affects its replication in the presence of cynomolgus monkey and human TRIM5alphas

Abstract

Human immunodeficiency virus type 2 (HIV-2) strains vary widely in their abilities to grow in Old World monkey (OWM) cells such as those of cynomolgus monkeys (CM). We evaluated eight HIV-2 isolates for their sensitivities to CM TRIM5alpha, an anti-HIV factor in OWM cells. We found that different HIV-2 isolates showed differences in their sensitivities to CM TRIM5alpha. Sequence analysis showed that TRIM5alpha-sensitive viruses had proline at the 120th position of the capsid protein (CA), whereas TRIM5alpha-resistant viruses had either alanine or glutamine. Mutagenesis studies indicated that the single amino acid at the 120th position indeed affected the sensitivity of the virus to CM TRIM5alpha.

FIG. 1.

Hut78 cells (10⁵) were infected with CM-TRIM5α-SeV (open circles), Hu-TRIM5α-SeV (filled triangles), or CM-SPRY(−)-SeV (open squares) at a multiplicity of infection of 10 PFU. The CM-SPRY(−)-SeV domain was generated by PCR amplification of the first-296-amino-acid region of CM TRIM5α. Nine hours after infection, cells were inoculated with eight HIV-2 isolates, GH123, UC12, UC1, UC14, UC2, UC7, 9429, and 12741. Culture supernatants were periodically assayed for levels of p25 by using a RETROtek antigen enzyme-linked immunosorbent assay kit (ZeptoMetrix Corp., Buffalo, NY). Error bars show actual fluctuations between measurements of p25 in duplicate samples. A representative of three independent experiments is shown.

FIG. 2.

(A) Alignments of amino acid sequences of CAs of eight HIV-2 isolates. “/S” after the names of isolates represents sensitivity to CM and Hu TRIM5α, and “/R” indicates resistance to CM and Hu TRIM5α. A dash denotes that the amino acid residue is identical to that of GH123. Blank spaces denote a lack of the amino acid residue that is present only in GH123. The box indicates the amino acid residues that correlate with susceptibility to restriction by CM and Hu TRIM5α. (B) Alignments of partial amino acid sequences of CA of all the HIV-2 and SIV isolates obtained from the Los Alamos database. A dash denotes that the amino acid is identical to that of HIV-2 UC2. X denotes amino acids unidentified due to sequence ambiguity. The box indicates the amino acid residues that correlate with susceptibility to restriction by CM and Hu TRIM5α.

FIG. 3.

(A) Mutant GH123/A and GH123/Q viruses generated by changing a single amino acid of proline to alanine or glutamine and mutant SIVmac239/P virus generated by changing a single amino acid of glutamine to proline by site-directed mutagenesis. Dashes indicate the unmutated amino acid residues. +++, ++, +, and − denote more-than-300-fold, 40- to 100-fold, 6- to 40-fold, and less-than-2.5-fold suppression of virus growth, respectively, compared with what was seen for the negative control at day 6 in the presence of the various TRIM5αs indicated. (B to D) MT4 cells were infected with CM-TRIM5α-SeV (open circles), Hu-TRIM5α-SeV (filled triangles), AGM-TRIM5α-SeV (filled squares), or CM-SPRY(−)-SeV (open squares). Nine hours after infection, cells were inoculated with GH123, mutant GH123/A, or GH123/Q viruses (panel B), with SIVmac239 or mutant SIVmac239/P virus (panel C), or with HIV-1 NL43 virus (panel D). Culture supernatants were periodically assayed for levels of virus CA. Error bars show actual fluctuations between measurements of CA in duplicate samples. A representative of three independent experiments is shown.

FIG. 4.

Structural model of the N-terminal halves of HIV-2 CAs. The 3-D models of 10 HIV-2 CAs were constructed with the homology-modeling technique using “MOE-Align” and “MOE-Homology” in the Molecular Operating Environment (MOE) as described previously (18, 19). Superimposition of the 10 HIV-2 models showed that the overall 3-D structures of the N-terminal domains of the variants are very similar. N and C indicate the amino termini and carboxyl termini, respectively. The ribbons represent the backbones of HIV-2 CAs, and the seven color-coded α-helices are labeled. P, A, and Q indicate L6/7 with a proline residue (GH123 and UC12 [in red]), an alanine residue (UC1, UC14, and GH123/A [in green]), and a glutamine residue (UC2, UC7, 9429, 12741, and GH123/Q [in blue]), respectively, at the 120th position. L4/5 is labeled with a color scheme the same as that for L6/7. The presence of proline residues conferred a sensitive phenotype; alanine and glutamine conferred phenotypes resistant to restriction by CM TRIM5α. Models from two different angles are shown.