Mechanism of HIV-1 capsid-nucleocapsid polyprotein in genome recognition (VIR9P.1143)

Abstract

Abstract Human Immunodeficiency Virus type-1 (HIV-1) is a retrovirus that is responsible for a widespread epidemic affecting nearly 36 million individuals worldwide. During the late phase of the viral life cycle, the Gag polyprotein specifically binds two copies of the unspliced viral genomic RNA. Gag consists of three structured domains: matrix (MA), capsid (CA), and nucleocapsid (NC). We have shown that mature NC binds to both the monomeric and dimeric 5’-untranslated region (5’-UTR) RNAs, but fails to explain the observation that the dimeric genome is preferentially packaged. Additionally, mature NC does not exist in the cytoplasm, where genome recognition occurs. Thus, this has led to the belief that other domains of Gag may also be involved in the selection of dimeric genomes. We hypothesize that the CA domain contributes to diploid RNA genome selection. In order to test this hypothesis, the polyprotein Capsid-Nucleocapsid (CA-NC) will be purified and titrated into 5’-UTR viral RNA. Previous work has shown that this CA-NC polyprotein is unstable and aggregates in the presence of RNA due to hexameric interactions mediated at the C-terminal domain (CTD). We will utilize the strategy of Gamble et al., by introducing two critical mutations, W184A and M185A, in the CTD domain of CA. These mutations have been shown to block CTD dimerization resulting in the monomeric form of the CA protein, while preserving necessary characteristics of the wild-type CA.