Structural Insights into Viral Membrane Fusion Machinery via Cryo-Electron Tomography: Influenza Virus and Human Parainfluenza Virus

Abstract

Enveloped viruses such as influenza virus and human parainfluenza virus utilize specialized protein machinery to fuse their membrane with the cellular membrane of target host cells and thus deliver their genome for replication. This protein-mediated membrane fusion is also a ubiquitous and key event that underlies many fundamental cellular processes. Despite its biological significance, the states that drive the fusion process have been refractory to classical structure determination, and the interplay of fusion proteins and membranes remains exclusive. My dissertation focused on direct structural characterization of viral fusion proteins and interplay between fusion proteins and membrane during fusion, using a combined approach of cryo-electron tomography (cryo-ET) and fluorescence spectroscopy. Firstly, in Chapter 2, I have investigated the 3-dimensional organization and population kinetics of the intermediates during fusion of influenza virus with membranes. I observed that progression of membrane reorganization proceeded through an extended contact zone with tightly apposed virus-target membrane interactions and this study provided the first demonstration of the sequence of membrane deformations during fusion. In Chapter 3, influenza virus fusion peptide-induced membrane deformation for isolated fusion proteins was compared with the behavior on whole viruses. I also examined the influence of cholesterol on the type of membrane deformations that were induced by activated HA. This study showed that isolated, soluble influenza virus fusion protein by itself could induce significant membrane deformation and that cholesterol had a very noticeable effect in stabilizing the target membrane against fusion protein-induced membrane deformation. Finally, I examined a parallel enveloped virus system to influenza virus by studying the organization of surface glycoproteins on human parainfluenza virus 3 (HPIV3) in Chapter 4. Using a combination of negative-staining and cryo-ET I was able to resolve the distributions of receptor binding protein and fusion protein, even to identify the conformational states of these proteins on the virus surface in some cases. My observations are consistent with a model for fusion in which prefusion fusion proteins associate with receptor binding proteins prior to receptor engagement. In conclusion, my studies have shown that a combination of biophysical and structural approaches can provide new insights into the process of protein-mediated membrane fusion.