Millions of people are currently infected with human immunodeficiency virus type 1 (HIV-1). HIV-1 is a retrovirus that attacks the CD4 cells of the immune system and integrates its proviral genome into the host genome to cause infection. Without proper treatment, the infection will progress to acquired immunodeficiency syndrome (AIDS). During the viral life cycle, viral assembly is initiated by interactions between unspliced viral RNA and its translated product, the Gag polyprotein. Gag is composed of three domains: Matrix (MA), Capsid (CA), and Nucleocapsid (NC). The NC domain binds to regions in the 5′ leader (5′-L) within the dimeric viral genome to initiate the genome packaging step of the HIV-1 replication cycle. This project seeks to characterize the Gag-RNA interactions vital to genome packaging by using a truncated 5′-L derivative and a hexameric NC protein. A hexamer of Gag is the basic unit of the immature viral shell. Previous studies have shown that mutations at the hexameric interface cause a significant reduction in Gag’s selectivity towards dimeric viral RNA. We hypothesize that the hexameric structure of CA is necessary in the selection of the dimeric RNA genome. However, hexamer-hexamer interactions in the C-terminal domain (CTD) of the CA domain lead to aggregation of protein and precipitation with RNA. To avoid this problem, we have fused the NC domain to a hexameric protein scaffold, which mimics the hexameric Gag structure, but forms hexamers that will not aggregate in solution. The truncated 5′-L contains only the regions required for NC binding and packaging. With these RNA and protein constructs, our goal is to deduce structural information and qualitative conditions of Gag-RNA interactions by using electrophoretic mobility shift assays, isothermal titration calorimetry, nuclear magnetic resonance, X-ray crystallography, and cryo-electron microscopy. These studies will further elucidate the mechanism of HIV-1 genome selection, which is a critical step of the life cycle that can be targeted with therapeutics.

Characterizing the interactions between the Gag polyprotein and dimeric RNA in HIV-1 viral assembly

254th National Meeting (2017)

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