Multimerization and Membrane Distribution of the Gag Structural Polyprotein During HIV-1 Assembly.

Abstract

Assembly of virus particles, a critical step in the HIV-1 replication cycle, is driven by the structural polyprotein Gag. Assembly is a complex process involving functionally interdependent molecular mechanisms: Gag binds to the plasma membrane via N-terminal myristoylation and basic residues that bind the phospholipid PI(4,5)P2. Gag multimerizes via dimerization of its capsid domain, and basic residues in its nucleocapsid domain that bind RNA as a scaffold. Gag complexes bud through the plasma membrane, forming a new virus particle. Yet, how these factors and functions interact in a complex system of interdependent molecular mechanisms remains less clear. The work presented in this dissertation focuses on unraveling the interrelationships between membrane binding, microdomain distribution, and multimerization of Gag during assembly. Using a microscopy-based assay of Gag multimerization in cells, we showed that capsid dimerization is essential for Gag multimerization, but nucleocapsid is only essential when Gag membrane binding is impaired. When Gag membrane binding is constitutively enhanced, nucleocapsid was completely dispensable. This interplay between membrane binding and nucleocapsid function suggests a model in which Gag binding to the plasma membrane and Gag binding to RNA via nucleocapsid play mechanistically similar roles – RNA and membrane may both serve as scaffolds to promote multimerization. Having demonstrated the importance of membrane binding for assembly, we next examined the relationship between different kinds of membrane microdomains. Previous studies proposed that lipid rafts and tetraspanin-enriched microdomains (TEM) are membrane platforms for HIV-1 assembly, although they are thought to be distinct from each other in the absence of HIV-1. Using an antibody-mediated co-patching assay, we showed that Gag causes the reorganization of membrane microdomains to coalesce lipid rafts and TEMs. Gag membrane binding was necessary, and alternative modes of membrane binding, either acylation or PI(4,5)P2 binding, each allow Gag-induced microdomain coalescence. This reorganization of microdomains may have important impacts on viral fitness. Currently, there are no antiretroviral drugs targeting HIV-1 assembly, but assembly is an attractive drug target because it is essential for virus replication. Thus, there is a great need for ongoing research to better understand the interrelated functions of Gag leading to assembly.