THE HARC CENTER: HIV ACCESSORY AND REGULATORY COMPLEXES PROJECT 1: STRUCTURE AND EVOLUTION OF THE VIF-APOBEC3 COMPLEX SUMMARY In Project 1 we will elucidate novel structural aspects of the APOBEC3 (A3) family of restriction factors and how they are antagonized by the HIV accessory protein Vif. Primate Vif targets A3’s for degradation by the 26S proteasome, but it is unknown how Vif intercepts A3 packaging complexes. It has been suggested that Vif binds different A3 family members through three different interfaces, but whether these binding sites are independent or dependent on one another is unclear. In previous studies, we uncovered that Vif forms functional interactions with additional host factors, including regulatory subunits of PP2A, components of the chromatin-modifying and transcriptional machinery, and regulators of ubiquitin-mediated proteolysis. We will now investigate how Vif neutralizes different A3 family members to promote efficient viral replication, and how adaptations in Vif enabled neutralization of A3G in hominid primates and how these adaptations affect the ability of human A3G to escape HIV-1. We will determine the structure of A3G and PP2A regulatory subunits bound to Vif using cryo-EM (Structural Biology Core), as well as deep mutational scanning (DMS) (Genetics Core), to uncover the mechanisms by which Vif recognizes different substrates and multitasks the degradation of A3 and PP2A subunits. The functional significance of structural observations will be further tested using viral and biochemical assays, and DMS in primary CD4+ T cells will explore tradeoffs between the ability of Vif to neutralize specific A3 family members vs. others. We will also use cryo-EM (Structural Biology Core), functional studies, and DMS (Genetics Core) to determine how Vif’s ability to engage restriction factors is rewired by adaptations allowing cross-species transmission. This is important because A3G and Vif undergo repeated bouts of positive selection and adaptation in what has been termed a ‘molecular arms race’, a process which led to cross-species transmission and the birth of HIV-1. Finally, we will investigate the mechanism of A3 packaging in the absence of Vif by determining composition and architecture of A3 packaging complexes, a long-standing question in the field. We will use the HEPS platform to discover host and viral proteins required for packaging of newly synthesized A3 family members (Proteomics Core). CRISPR-Cas9 and mutagenesis will determine the functional significance of the A3 packaging complex (Genetics Core). Cryo-EM studies will be performed on the packaging complex (Structural Biology Core). These approaches will provide snapshots of A3 family members en route to packaging and define how Vif intercepts these structures to promote viral infectivity. Discoveries made by Project 1 will enable rational drug design to target HIV-1 from establishing replication-competent proviruses by utilizing the restriction potential...