Title: Stabilization of Human Immunodefiency Virus 1 envelope glycoprotein conformations
Description: Human immunodeficiency virus-1 (HIV-1) is composed of multiple functional units held together by weak interactions. Some of these interactions involve conformational flexibility within viral protein components that is important for the biological function of the virus. Such interactions may represent targets for disruption by small-molecule drugs. We attempted to gain insight into these interactions by subjecting virus to heat and cloning temperature-resistant isolates. One of the key functional complexes of the virus is the envelope glycoprotein trimer, which is composed of gp120 and gp41 subunits. Upon binding to the receptors, CD4 and CCR5/CXCR4, the envelope glycoproteins will undergo a series of conformational changes that promote virus entry. In this dissertation, we report the generation of temperature-resistant HIV-1 and the identification of a single change (histidine 66 (H66) to asparagine (N)) that was responsible for the heat-resistant phenotype. The H66N mutation stabilized the native form of the envelope glycoprotein by preventing spontaneous sampling of the labile CD4-bound conformation. Once the enevelope glycoprotein bound to CD4, the H66N change stabilized the functional intermediate formed, distinguishing for the first time the activation versus inactivation pathway in HIV-1 entry. In the CD4-bound state, H66 is strategically located at the interface of gp120 and gp41 subunits, controlling conformational transitions of the envelope glycoprotein in response to receptor binding. This enabled the H66N change to exert a striking effect on the susceptibility of HIV-1 to drug inhibition and antibody neutralization.

In this dissertation, we also report a new temperature-dependent inactivation pathway of HIV-1. Surprisingly, we found that the envelope glycoproteins of a variety of HIV-1 strains are functionally inactivated by prolonged incubation on ice. However, the envelope glycoproteins of the selected heat-resistant virus also resisted cold inactivation again as a result of the H66N change. In attempting to find a structural basis for this phenotype, we found that sensitivity to cold inactivation is dependent on the degree of exposure of gp41 epitopes in the trirner. Induction of the CD4-bound conformation by a mutation in gp120 (S375W) or treatment with soluble CD4 or a small-molecule CD4 mimetic resulted in increased cold sensitivity. These results indicate that the CD4-bound intermediate of HIV-1 envelope glycoproteins is particularly labile in the presence of destabilizing influences, such as heat or cold. Our results provide insight into the overall organization, conformational transitions, and the vulnerabilities of the HIV-1 envelope glycoprotein trimer.