Background:  The HIV Vif protein is essential for production of infectious virus.  Vif counteracts the innate antiviral activity of APOBEC3G, a cytidine deaminase that induces massive G to A hypermutation in the viral genome. In the absence of Vif, APOBEC3G incorporation into virions renders HIV non-infectious. Here, we investigate the mechanism by which Vif counteracts APOBEC3G.

Methods:  A Vif-APOBEC3G complex was demonstrated by reciprocal coimmunoprecipitation.  The effect of Vif on APOBEC3G steady-state levels was examined in transfected 293T and infected T cells. The half-lives of APOBEC3G and Vif were determined by pulse-chase experiments. Protein ubiquitination and proteasome-mediated degradation were investigated using epitope-tagged and mutant forms of ubiquitin (Ub_K48R) and proteasome inhibitors.

Results:  Vif forms a complex with APOBEC3G and enhances APOBEC3G ubiquitination, resulting in reduced steady-state APOBEC3G levels and a decrease in protein half-life. Furthermore, Vif-dependent degradation of APOBEC3G is blocked by proteasome inhibitors or ubiquitin mutant K48R. Mutation of highly conserved cysteines in Vif results in a mutant that fails to induce APOBEC3G degradation and results in non-infectious HIV. Surprisingly, the stability of Vif is also dependent on APOBEC3G. Vif is mono-ubiquitinated in the absence of APOBEC3G, but is polyubiquitinated and rapidly degraded when APOBEC3G is co-expressed, suggesting that coexpression accelerates the degradation of both proteins. The reduced half-lives of Vif and APOBEC3G in the presence of each other are similar, i.e. around 23 minutes and 18 minutes, respectively, suggesting that these 2 proteins may be coordinately degraded as a complex. A subpopulation of APOBEC3G appears to be resistant to Vif-dependent degradation.  Cellular factors that regulate Vif-dependent degradation of APOBEC3G have been identified.

Conclusions:  These results suggest that Vif has evolved to target APOBEC3G for degradation via the ubiquitin-proteasome pathway, and support a model in which Vif functions as an adaptor molecule, recruiting APOBEC3G to the ubiquitin-proteasome machinery, but at the cost of its own destruction. The interaction between the ubiquitin machinery and the Vif-APOBEC3G complex implicates the proteasome as a site of dynamic interplay between viral and host defenses, and provides a new interface for pharmaceutical intervention.

Keywords: Vif; APOBEC3G; proteasome