Potent inhibition of HIV-1 by TRIM5-cyclophilin fusion proteins engineered from human components

Abstract

New World monkeys of the genus Aotus synthesize a fusion protein (AoT5Cyp) containing tripartite motif-containing 5 (TRIM5) and cyclophilin A (CypA) that potently blocks HIV-1 infection. We attempted to generate a human HIV-1 inhibitor modeled after AoT5Cyp, by fusing human CypA to human TRIM5 (hT5Cyp). Of 13 constructs, 3 showed substantial HIV-1-inhibitory activity when expressed in human cell lines. This activity required capsid binding by CypA and correlated with CypA linkage to the TRIM5a capsid-specificity determinant and the ability to form cytoplasmic bodies. CXCR4- and CCR5-tropic HIV-1 clones and primary isolates were inhibited from infecting multiple human macrophage and T cell lines and primary cells by hT5Cyp, as were HIV-2ROD, SIVAGMtan, FIVPET, and a circulating HIV-1 isolate previously reported to be AoT5Cyp resistant. The anti-HIV-1 activity of hT5Cyp was surprisingly more effective than that of the well-characterized rhesus TRIM5alpha, especially in T cells. hT5Cyp also blocked HIV-1 infection of primary CD4+ T cells and macrophages and conferred a survival advantage to these cells without disrupting their function. Extensive attempts to elicit HIV-1 resistance to hT5Cyp were unsuccessful. Finally, Rag2-/-gammac-/- mice were engrafted with human CD4+ T cells that had been transduced by optimized lentiviral vectors bearing hT5Cyp. Upon challenge with HIV-1, these mice showed decreased viremia and productive infection in lymphoid organs and preserved numbers of human CD4+ T cells. We conclude that hT5Cyp is an extraordinarily robust inhibitor of HIV-1 replication and a promising anti-HIV-1 gene therapy candidate.

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

“…Both are predicted to show low immunogenicity when used in vivo. hT5cyp probably has higher potency, 56 certainly not a negligible factor. The single most important determinant for success in vivo, however, is likely to be the emergence (or lack thereof) of HIV-1 variants resistant to the transgenic restriction factor used.…”

“…Our mutant TRIM5a hu antiviral approach begs the comparison with hT5Cyp, 56 the recently constructed TRIMCyp-like restriction factor with very high HIV-1 restriction activity. Both types of gene are likely to inhibit HIV-1 through the same effector mechanisms, although that remains to be assessed.…”

Birth, decay, and reconstruction of an ancient TRIMCyp gene fusion in primate genomes

“…We also designed a chimera composed of owl monkey TRIM5 effector domains and the inferred parental CypA gene (TRIM5-parentalCypA), representing the CypA gene from which CypA3 derived at the time of its birth. Because CypA genes have been evolving under strong purifying selection throughout primate history, this approach allows us to evaluate the lentiviral specificity of a de novo CypA retrogene unambiguously, representing TRIMCypA3 immediately following its birth (36). Consistent with previous results, we observed that owl monkey TRIMCypA1 was not able to restrict HIV-2 (4) but was able to restrict HIV-1 (15), simian immunodeficiency virus from african green monkeys (SIVagm) (18), and feline immunodeficiency virus (FIV) (37), as well as chimeric viruses encoding the reconstructed capsid of the "paleoviruses" RELIK and pSIV (38) (Fig.…”

A Mutant Tat Protein Provides Strong Protection from HIV-1 Infection in Human CD4+T Cells

“…RevM10 mutant, the first trans-dominant negative protein to undergo a pilot clinical trial in HIV-1-infected patients (Woffendin et al, 1996;Ranga et al, 1998), was beneficial to cell survival in the context of HIV infection (Podsakoff et al, 2005). A human engineered TRIM5 (tripartite motif 5)-cyclophilin protein introduced into primary CD4 + T cells and macrophages provided extraordinary protection from HIV-1 infection in vitro and in immunecompromised mice (Neagu et al, 2009). TRIM5-cyclophilin inhibits HIV-1 infection in the newly infected cell by binding to capsid protein (CA), leading to downregulated viral reverse transcription.…”