A single amino acid of human immunodeficiency virus type 2 capsid protein affects conformation of two external loops and viral sensitivity to TRIM5α

Abstract

We previously reported that human immunodeficiency virus type 2 (HIV-2) carrying alanine or glutamine but not proline at position 120 of the capsid protein (CA) could grow in the presence of anti-viral factor TRIM5α of cynomolgus monkey (CM). To elucidate details of the interaction between the CA and TRIM5α, we generated mutant HIV-2 viruses, each carrying one of the remaining 17 possible amino acid residues, and examined their sensitivity to CM TRIM5α-mediated restriction. Results showed that hydrophobic residues or those with ring structures were associated with sensitivity, while those with small side chains or amide groups conferred resistance. Molecular dynamics simulation study revealed a structural basis for the differential TRIM5α sensitivities. The mutations at position 120 in the loop between helices 6 and 7 (L6/7) affected conformation of the neighboring loop between helices 4 and 5 (L4/5), and sensitive viruses had a common L4/5 conformation. In addition, the common L4/5 structures of the sensitive viruses were associated with a decreased probability of hydrogen bond formation between the 97th aspartic acid in L4/5 and the 119th arginine in L6/7. When we introduced aspartic acid-to-alanine substitution at position 97 (D97A) of the resistant virus carrying glutamine at position 120 to disrupt hydrogen bond formation, the resultant virus became moderately sensitive. Interestingly, the virus carrying glutamic acid at position 120 showed resistance, while its predicted L4/5 conformation was similar to those of sensitive viruses. The D97A substitution failed to alter the resistance of this particular virus, indicating that the 120th amino acid residue itself is also involved in sensitivity regardless of the L4/5 conformation. These results suggested that a hydrogen bond between the L4/5 and L6/7 modulates the overall structure of the exposed surface of the CA, but the amino acid residue at position 120 is also directly involved in CM TRIM5α recognition.

Figure 1. Growth of GH123 and its mutant viruses in the presence of CM TRIM5α.

MT4 cells were infected with CM-TRIM5α-SeV (black circles) or CM-SPRY(–)-SeV (white circles) then superinfected with GH123 mutant viruses. Culture supernatants were periodically assayed for levels of virus capsid. Error bars show actual fluctuations between measurements of capsid in duplicate samples. A representative of two independent experiments is shown.

Figure 2. Western blot analysis of the CA in particles of GH123 and its mutant viruses.

The viral particles of GH123 wild type and its mutant viruses were purified by ultracentrifugation through a 20% sucrose cushion. p25 capsid protein was visualized by western blotting (WB) using SIV-infected monkey serum.

Figure 3. Growth of SIVmac239 and its mutant viruses in the presence of CM TRIM5α.

MT4 cells were infected with CM-TRIM5α-SeV (black circles) or CM-SPRY(–)-SeV (white circles) then superinfected with SIVmac239 mutant viruses. Culture supernatants were periodically assayed for levels of virus capsid. Error bars show actual fluctuations between measurements of capsid in duplicate samples. A representative of three independent experiments is shown.

Figure 4. Structural models of the HIV-2 capsid N-terminal domain.

Models were constructed by homology modeling and molecular dynamics simulations with the high-resolution X-ray crystal structure of the HIV-2 capsid N-terminal domain (PDB code: 2WLV [29]) as the starting structure. Averaged conformations of the overall structure of the N-terminal domain during 5–20 nanoseconds of MD simulations (A and B) and a close-up view around the L4/5 loop (C and D) are indicated. N and C indicate the amino termini and carboxyl termini, respectively; and the seven color-coded α-helices are labeled. Red and blue cartoons indicate the N-terminal loop, L4/5, and L6/7 of CM TRIM5α-sensitive (GH123/P, GH123/F, GH123/H and GH123/I) and CM TRIM5α-resistant (GH123/Q, GH123/A and GH123/N) viruses, respectively. Gray cartoons indicate the N-terminal loop, L4/5 and L6/7 of GH123/E in which the structures and biologic phenotypes are inconsistent. Models from two different angles are shown.

Figure 5. Hydrogen bond formation among L4/5, L6/7 and helix 6 of the HIV-2 CA.

Close-up views of averaged structures of the N-terminal domain of the GH123/P CA during 5–20 nanoseconds of MD simulations are shown. Red, blue, purple, orange and green wireframes denote side chains of arginine at the 96th (96R), aspartic acid at the 97th (97D), glutamic acid at the 112th (112E), tryptophan at the 116th (116W) and arginine at the 119th (119R) positions, respectively. Dotted lines indicate hydrogen bonds visualized with MOE 2009. Models from two different angles are shown.

Figure 6. Lack of hydrogen bond formation between the 97th alanine and the 119th arginine of HIV-2 D97A-GH123/Q CA.

Close-up views of averaged structures around the L4/5 loop of GH123/Q (left) and D97A-GH123/Q (right) during 5–20 nanoseconds of MD simulations are shown. Red, blue and green wireframes denote side chains of aspartic acid at the 97th (97D), arginine at the 119th (119R), and alanine at the 97th (97A) positions, respectively. A dotted line indicates a hydrogen bond.

Figure 7. Effects of an aspartic acid-to-alanine substitution at the 97th position of the HIV-2 CA on viral growth in the presence or absence of CM TRIM5α.

MT4 cells were infected with CM-TRIM5α-SeV (black circles) or CM-SPRY(–)-SeV (white circles) then superinfected with GH123 mutant viruses. Culture supernatants were periodically assayed for levels of viral capsid. Error bars show actual fluctuations between measurements of capsid in duplicate samples. A representative of three independent experiments is shown.

Figure 8. Surface structure of the HIV-2 capsid N-terminal domain.

Surface structure of the GH123 and mutant GH123 CAs visualized with PyMOL. Red color indicates the 120th amino acid of the GH123 and mutant GH123 CAs.