Viral Hijacking of Host E3 Ubiquitin Ligase Machinery for the Degradation of Host Antiviral Factors

The HIV-1 virion infectivity factor (Vif) targets the cellular cytidine deaminase, A3G, for degradation by the host proteasome to enable the virus to carry out a productive infection. Vif helps HIV-1 evade the host innate immune response by hijacking a cellular E3 ubiquitin ligase composed of Cullin 5 (Cu15), ElonginB and ElonginC (EloBC). Vif mimics a host SOCS box protein, the substrate receptor component in the ligase, to accomplish this assembly. To delineate the molecular mechanism of how Vif hijacks the host ubiquitylation pathway we have probed the assembly of the ligase through biochemical and biophysical studies. We have developed a strategy to express and purify various subcomplexes of the Vif-E3 ligase, which has allowed us to not only obtain complexes containing up to five components of the ligase for biochemical studies and crystallization trials but also enabled us to explore and characterize ligase assembly in vitro. Furthermore, we have probed the selective binding of Vif to Cul5 over other cullin family members through mutational studies. The results of this work have provided insight into how HIV-1 counteracts the host immune system through selectively assembling a Cul5-based E3 ligase. The results also have paved the way for future studies by overcoming solubility issues of many components of the ligase.;Additionally, we have investigated the effects of the newly identified Vif-binding factor, the core binding factor β (CBFβ), on ligase stability and assembly. While the exact role of CBFβ in Vif-E3 ligase function is not fully understood, it has been shown that CBFβ is required for Vif stabilization and for subsequent A3G degradation in vivo. Co-expression of Vif with CBFβ results in a behaving Vif in solution, a feat previously impossible due to the insolubility of full-length Vif. The addition of CBFβ to our repertoire of Vif binding partners has opened a door to probe the mechanisms of this crucial cellular factor as well as has expanded our abilities to obtain Vif-E3 ligase subcomplexes.;Finally, we have developed a high-throughput screening assay to identify small molecule compounds that inhibit Vif-E3 ligase assembly. More specifically, we have screened 14,014 compounds for disruption of the Vif-Cul5 interface and 2,880 compounds to test for inhibition of Vif-CBFβ binding. We have identified lead compounds and confirmed them through dose response assays and biochemical experiments. Taken together, our studies on the Vif-E3 ligase complex have expanded our understanding of the mechanism by which HIV-1 utilizes Vif to target the important cellular antiviral factor, A3G, for degradation, laid the groundwork for future in vitro studies of the Vif-E3 ligase complex, and established a strategy to identify Vif inhibitors that may lead to potential functional probes and new anti-HIV therapeutics.