Characterizing Protein and RNA Interactions that Nucleate the HIV-1 Viral Assembly

Abstract

Human immunodeficiency virus type 1 (HIV-1) has become a worldwide epidemic. During the HIV life cycle, the viral assembly is initiated by interaction between the unspliced viral genomic RNA (vRNA) and its translated product, the Gag polyprotein. Although there is a large excess of non-viral RNA, Gag protein is able to efficiently package the vRNA. Gag contains three domains: Matrix (MA), Capsid (CA), and Nucleocapsid (NC). The CA domain mediates Gag-Gag interaction resulting in the hexagonal Gag shell that encapsulates the viral RNA while the NC domain of Gag recognizes exposed guanosines in the 5′-leader (5′-L) region of the vRNA genome for selective packaging. We seek to characterize the Gag-RNA interactions essential to genome packaging using a truncated 5′-L derivative and NC protein constructs. We hypothesize that the hexameric structure of the CA domain contributes to the RNA genome selection. The basic unit of the immature viral shell is a hexamer of Gag; mutations at the hexameric interface significantly reduce Gag's selectivity towards dimeric viral RNA. Using Nuclear Magnetic Resonance (NMR), Isothermal Titration Calorimetry (ITC) and electrophoretic mobility shift assays (EMSA) about sixteen binding sites for Gag proteins on the core encapsidation signal (CES) have been identified which promote the formation of a Gag hexamer. This protein-RNA initiation complex is suggested to function as the nucleation site to recruit more Gag proteins. Other techniques as cryo-electron microscopy are used to study the structural information about this initiation complex. We aim to solve the structure of this protein-RNA initiation complex which will provide the detailed molecular mechanism for selective genome packaging. These studies will further our understanding of the mechanism of HIV genome selection, a stage in the life cycle that can be targeted with therapeutics.