Background:  HIV-1 mutation drives viral escape from immune responses and antiretroviral therapy. APOBEC-3G (h3G), a host cell cytodine deaminase, induces such extensive viral hypermutation that replication is severely attenuated in the absence of vif. The goal of this study was to identify which APOBEC contributes to viral evolution by inducing non-lethal mutations in a vif-independent manner. APOBEC-3C (h3C), expressed in PBMC, causes G to A hypermutation in a GA context and may facilitate vif-independent HIV-1 hypermutation and accelerated evolution in vivo.

Methods:  HIV-1 virus stocks (NL4-3, NL4-3 Dvif, and a molecular clone of patient-derived gag and pro in an isogenic backgound) were generated in 293T cells. Single-round and short-term infections in PBMC and cell lines were monitored by soluble p24. Viral hypermutation was detected by population sequencing of proviral DNA using hypermutated published primers. h3C was cloned from H9 cells. Endogenous h3C expression was downregulated in cell lines using RNAi.

Results:  We found evidence of viral hypermutation in some, but not all, cell lines and PBMC infected with the patient-derived molecular clone. In cells that express h3G (PBMC, H9), G-A viral hypermutation occurred in a GG context following infection with NL4-3 Dvif. In cells expressing h3C, G-A hypermutation occurred primarily in the GA context, was independent of vif expression, and occurred primarily with the patient-derived molecular clone. Viral hypermutation was absent in cells lacking h3C and h3G. Transient expression of h3C led to HIV-1 hypermutation in h3C null cells. Inhibition of endogenous h3C using specific RNAi abrogated viral hypermutation in two cell lines. Hypermutation caused by h3C is not lethal for viral replication.

Conclusion:  h3C is necessary and sufficient for hypermutation of a patient-derived HIV-1 infectious molecular clone. In contrast with h3G, the action of h3C is non-lethal and is not suppressed by Vif, although there appear to be other viral determinants that modify h3C action. RNAi inhibition of h3C decreases viral mutation in cell culture. Inhibition of h3C may represent a novel strategy for delaying viral escape from immune or antiretroviral drug inhibition.

Keywords: APOBEC3C; Hypermutation; Viral Evolution