A novel aminoacid determinant of HIV-1 restriction in the TRIM5α variable 1 region isolated in a random mutagenic screen

Abstract

Human-derived antiretroviral transgenes are of great biomedical interest and are actively pursued. HIV-1 is efficiently inhibited at post-entry, pre-integration replication stages by point mutations in the variable region 1 (v1) of the human restriction factor TRIM5α. Here we use a mutated megaprimer approach to create a mutant library of TRIM5αHu v1 and to isolate a mutation at Gly330 (G330E) that inhibits transduction of an HIV-1 vector as efficiently as the previously described mutants at positions Arg332 and Arg335. As was the case for these other mutations, modification of the local v1 charge toward increased acidity was key to inhibiting HIV-1. G330E TRIM5αHu also disrupted replication-competent HIV-1 propagation in a human T cell line. Interestingly, G330E did not enhance restriction of HIV-1 when combined with mutations at Arg332 or Arg335. Accordingly, the triple mutant G330E-R332G–R335G bound purified recombinant HIV-1 capsid tubes less efficiently than the double mutant R332G–R335G did. In a structural model of the TRIM5αHu PRYSPRY domain, the addition of G330E to the double mutant R332G–R335G caused extensive changes to the capsid-binding surface, which may explain why the triple mutant was no more restrictive than the double mutant. The HIV-1 inhibitory potential of Gly330 mutants was not predicted by examination of natural TRIM5α orthologs that are known to strongly inhibit HIV-1. This work underlines the potential of random mutagenesis to isolate novel variants of human proteins with antiviral properties.

Preclinical Assessment of Mutant Human TRIM5α as an Anti-HIV-1 Transgene

“…These results confirm our previously published data obtained in single-cycle infection experiments using HIV-1 vectors 23,29,31 or in spreading infection of a different HIV-1 strain (NL4-3) in a different T-cell line (Sup-T1). 23,31 Collectively, our data also show that, in lymphocytes, R332G-R335G TRIM5a hu restricts HIV-1 about as efficiently as TRIM5a Rh does.…”

“…In addition, the percentage of GFP-positive cells was proportional to the level of protection conferred by the respective TRIM5a variants: TRIM5a Rh > R332G-R335G TRIM5a hu > R332G TRIM5a hu . 23,29,31 Although the exact proportion of TRIM5a-expressing cells that survived HIV-1 infection in the first 4 weeks of infection is not known, this experiment suggests that R332G-R335G TRIM5a hu can confer protection not only against HIV-1 infection but also against cytopathic effects stemming from the active spread of the virus in the unmodified cells.…”

“…29,31,54 To produce Gag variant HIV-1 TRIP-CMV-GFP , the cells were cotransfected with pTRIP CMV-GFP , the respective pCMV-D8.91 vector, and pMD-G. To generate the gag variants of HIV-1 NL-Luc , the cells were cotransfected with the respective pNL4-3XCS vector and pMD-G. HIV-1 NL43 was produced by transfection of pNL4-3. HIV-1 IIIB was amplified by passage in stimulated peripheral blood mononuclear cells and was titered by end-point dilution in MAGI cells, as described before.…”

“…27 Modeling studies and genetic screens have also led to the identification of point mutations in the variable region 1 (v1) of the TRIM5a hu PRYSPRY domain that allow it to target HIV-1 for restriction. 23,[28][29][30] We previously described the R332G-R335G TRIM5a hu mutant as especially efficient at restricting HIV-1. In particular, this double mutant had significantly superior anti-HIV-1 activity compared with the previously described single mutation at position 332.…”

“…In particular, this double mutant had significantly superior anti-HIV-1 activity compared with the previously described single mutation at position 332. 23,29,31 One major limitation of gene therapy applications is the immune response that often results from introducing foreign proteins into humans. [32][33][34] Because the TRIM5a hu mutants differ only slightly from the endogenous form of TRIM5a hu , they are not expected to be immunogenic, thus making them strong candidates for gene therapy applications compared with simian orthologs.…”