| The Vif protein of human immunodeficiency virus 1 (HIV-1) acts by overcoming the antiviral activity of APOBEC3G, a cellular cytidine deaminase that mutates newly synthesized viral DNA. In the absence of Vif, APOBEC3G incorporation into virions renders HIV-1 non-infectious. Vif forms a complex with APOBEC3G and prevents its encapsidation into virions, thereby protecting the viral genome from editing mutations, but the mechanisms by which Vif inhibits APOBEC3G encapsidation are not well understood. This thesis addresses how Vif exploits the cellular ubiquitin machinery to overcome the innate antiviral activity of APOBEC3G. We demonstrated that Vif targets APOBEC3G for destruction by the ubiquitin-proteasome pathway. We showed that Vif binds directly to APOBEC3G and enhances APOBEC3G ubiquitination, resulting in reduced steady-state APOBEC3G levels, a decreased protein half-life, and enhanced viral infectivity. These results suggest that the primary function of Vif is to target APOBEC3G for proteasomal degradation. We then identified Vif-associated cellular proteins that mediate APOBEC3G ubiquitination, including Cullin 5 (Cul5), Elongin B (EloB), and Elongin C (EloC), which are core subunits of a Skp1-Cullin-F-box (SCF)-like E3 ubiquitin ligase complex. Vif bound directly to EloC via a highly conserved motif with homology to the SOCS (s&barbelow;uppressor o&barbelow;f c&barbelow;ytokine s&barbelow;ignaling)-box motif, a domain found in proteins that bind to EloC and function as specificity modules within Cul5 E3 ubiquitin ligases. Vif binding to EloC was negatively regulated by phosphorylation in the SOCS-box. Vif was shown to be autoubiquitinated by Cul5, analogous to F-box proteins that are autoubiquitinated within their SCF complex. These findings suggest that Vif mediates APOBEC3G degradation via a Vif-Cul5 complex and suggest mechanisms that regulate the assembly and activity of Cul5 SCF complexes. We then performed experiments to investigate the role of the novel conserved HX5C-X17--18-CX5H (HCCH) motif in Vif, which is similar to zinc coordinating sites found in metalloproteins. We showed that Vif is a zinc-binding protein by zinc-blot analysis, atomic absorption spectroscopy, and inductive-coupled plasma mass spectrometry. Mutation of the HCCH motif prevented Vif-dependent enhancement of viral infectivity, suggesting that zinc-binding is important for Vif function. We propose that the HCCH motif represents a novel zinc binding domain that stabilizes structural elements in Vif, similar to the role of zinc binding domains in cellular proteins. Thus, Vif is a zinc metalloprotein that targets APOBEC3G for degradation via a Vif-Cul5 E3 ubiquitin ligase complex. These findings provide new insights into the mechanisms by which Vif counteracts the antiviral activity of APOBEC3G through the cellular ubiquitin machinery, and also have significant implications for identifying new therapeutic targets. |
