Book Description

Protein-mediated membrane fusion is a highly regulated biological process essential for cellular and organismal functions and infection by enveloped viruses. During viral entry, the membrane fusion reaction is catalyzed by specialized protein machinery on the viral surface. These viral fusion proteins undergo a series of dramatic structural changes during membrane fusion where they engage, remodel, and ultimately fuse with the host membrane. The structural and dynamic nature of these conformational changes and their impact on the membranes have long-eluded characterization. Furthermore, the native pre-fusion structural and conformational dynamics of these fusion machines remains unclear as the conventional structural approaches employed by structural biologists are not well suited for studying these dynamic protein machines on the viral surface. The objective of this dissertation is to characterize the complete mechanism of Influenza virus hemagglutinin (HA) fusion activation and membrane fusion, and to profile and characterize the structural and conformational dynamics of the HIV-1 Env fusion glycoprotein on the viral surface. In chapter 2 I use continuous labeling HDX-MS to characterize the structural dynamics and conformational homogeneity of the HIV-1 Env fusion glycoprotein on the surface of two distinct engineered and authentic viral vaccine platforms. By HDX-MS we observed significant amounts of non-native Env present in one vaccine platform, whereas all Env present in the other resembled trimeric Env in the closed conformation. In chapter 3, I use pulse labeling HDX-MS to characterize the mechanism of HA fusion activation and HA mediated membrane fusion in situ using whole infectious virions. Our data reveal how concurrent reorganizations at the HA1 receptor binding domain interface and HA2 fusion subunit produce a dynamic fusion intermediate ensemble in full-length HA. In contrast, the soluble HA ectodomain transitions directly to the post-fusion state with no observable intermediate. These data provide unprecedented insight into the structural mechanics of HA which has served as the prototypical class 1 viral fusion protein and informed our understanding about how all class 1 viral fusion proteins function. In chapter 4 I present developments and improvements on the HDX-MS workflows that will enable more complete characterizations of HA's mechanism and the structural and conformational dynamics of other class 1 viral fusion proteins. Together these works have dramatically furthered our understanding of the structural mechanics of class 1 fusion proteins and lay the foundation for future studies on influenza virus and other enveloped viruses and their membrane fusion machinery.