Removal of Arginine 332 Allows Human TRIM5α To Bind Human Immunodeficiency Virus Capsids and To Restrict Infection

Abstract

Human TRIM5␣ (TRIM5␣ hu ) only modestly inhibits human immunodeficiency virus type 1 (HIV-1) and does not inhibit simian immunodeficiency virus (SIV mac ). Alteration of arginine 332 in the TRIM5␣ hu B30.2 domain to proline, the residue found in rhesus monkey TRIM5␣, has been shown to create a potent restricting factor for both HIV-1 and SIV mac. Here we demonstrate that the potentiation of HIV-1 inhibition results from the removal of a positively charged residue at position 332 of TRIM5␣ hu. The increase in restricting activity correlated with an increase in the ability of TRIM5␣ hu mutants lacking arginine 332 to bind HIV-1 capsid complexes. A change in the cyclophilin A-binding loop of the HIV-1 capsid decreased TRIM5␣ hu R332P binding and allowed escape from restriction. The ability of TRIM5␣ hu to restrict SIV mac could be disrupted by the presence of any charged residue at position 332. Thus, charged residues in the v1 region of the TRIM5␣ hu B30.2 domain can modulate capsid binding and restriction potency. Therapeutic strategies designed to neutralize arginine 332 of TRIM5␣ hu might potentiate the innate resistance of human cells to HIV-1 infection.Primates express dominant restriction factors that block retrovirus infection soon after entry but prior to reverse transcription (1, 2, 5). Genetic studies of virus variants and restriction factor competition studies indicate that the viral capsid is the determinant of susceptibility to restriction (3, 3b, 6, 13, 14). Most early restriction in primates is mediated by TRIM5␣ (7,10,16,23,26). TRIM5␣ is a member of the tripartite motif family of proteins and contains RING, B-box 2, and coiled-coil (RBCC) domains (17). TRIM5␣ also contains a C-terminal B30.2/SPRY domain, which is required for retroviral restriction (23). Deletion of the B30.2 domain disrupts the ability of the TRIM5␣ protein to bind viral capsid complexes (20,24). Differences in the B30.2 domains of TRIM5␣ proteins from distinct primate species account for patterns of retrovirus restriction. For example, the rhesus monkey TRIM5␣ (TRIM5␣ rh ) potently blocks human immunodeficiency virus type 1 (HIV-1), which is only weakly inhibited by human TRIM5␣ (TRIM5␣ hu ) (23). Neither TRIM5␣ rh nor TRIM5␣ hu efficiently restricts simian immunodeficiency virus (SIV mac ) (23). Four variable regions (v1 to v4) are found in the B30.2 domains of TRIM5␣ proteins from different primates (19,21). Differences in the v1 regions of TRIM5␣ rh and TRIM5␣ hu account for the differences in anti-HIV-1 potency of these TRIM5␣ variants (15,19,24,27). Alteration of arginine 332 in the v1 region of TRIM5␣ hu to the proline residue found in TRIM5␣ rh results in a protein that can potently restrict HIV-1 and, surprisingly, SIV mac infection (24, 27). Here we investigate the specific v1 sequences in TRIM5␣ hu required for efficient antiviral activity against HIV-1 and SIV mac and provide a mechanistic explanation for the observed enhancement of restriction that results from changes in this region. MATERIALS AND METHODS Plasmids a...

The structural biology of HIV assembly

“…TRIM5α restriction is species-and virus-specific, and the specificity of restriction is largely determined by interactions between the C-terminal SPRY/B30.2 domain and the intact retroviral capsid. Although structural details are still lacking, mutational studies and sequence analyses of different SPRY alleles have provided insights into the determinants of capsid recognition (e.g., see [138][139][140][141]). In TRIM-Cyp, the SPRY domain of TRIM5α is replaced by cyclophilin A (CypA), a well-known peptidyl prolyl isomerase.…”

Generation of human TRIM5α mutants with high HIV-1 restriction activity

“…For instance, TRIM5a rh with an arginine at this position instead of a leucine was still fully able to restrict HIV-1. 35 Li et al 36 constructed the reciprocate R335L mutation of TRIM5a hu and found that it weakly inhibited HIV-1; in addition, the double-mutant R332P/ R335L restricted HIV-1 less efficiently than R332P alone. Therefore, isolation of an Arg335 mutant in our screen was rather unexpected.…”

“…36 Although the protection provided by mutations at position 332 was strong (10-to 30-fold), these mutants still restricted HIV-1 10-times less efficiently than TRIM5a rh did. 36 Numerous gene therapy clinical trials for the treatment of AIDS have been undertaken to mostly disappointing results, both in terms of viral load and of CD4+ T cells counts. 38 In particular, although it is desirable that transgene-expressing cells survive and proliferate in the recipient patients, the opposite is often observed.…”

“…33,35 In particular, substitution of TRIM5a hu arginine 332 for a proline (the aminoacid present at that position in TRIM5a rh ) decreased permissiveness to HIV-1 infection. [35][36][37] In fact, most substitutions of this residue, excepted to a lysine, resulted in significant HIV-1 restriction, showing that suppression of the positive charge was the mechanism underlying the acquisition of HIV-1 restriction potential. 36 Although the protection provided by mutations at position 332 was strong (10-to 30-fold), these mutants still restricted HIV-1 10-times less efficiently than TRIM5a rh did.…”

“…[35][36][37] In fact, most substitutions of this residue, excepted to a lysine, resulted in significant HIV-1 restriction, showing that suppression of the positive charge was the mechanism underlying the acquisition of HIV-1 restriction potential. 36 Although the protection provided by mutations at position 332 was strong (10-to 30-fold), these mutants still restricted HIV-1 10-times less efficiently than TRIM5a rh did. 36 Numerous gene therapy clinical trials for the treatment of AIDS have been undertaken to mostly disappointing results, both in terms of viral load and of CD4+ T cells counts.…”