Biophysical studies of viral membrane fusion proteins

SUMMARY Influenza A virus (IAV) hemagglutinin (HA) is the canonical example of a class-I viral fusion protein, and thus provides an ideal model system for understanding the fusion mechanisms of many different viruses. Numerous other viral envelope glycoproteins, including the SARS-CoV-2 spike, are believed to mediate fusion by a comparable mechanism. Our long-term goal is to establish a complete mechanistic framework of class-I viral fusion. We further aim to identify conserved and divergent features of the fusion mechanisms of distinct viruses, generating specific models that fit within the general framework. HA resides on the surface of the IAV virion and facilitates attachment to the target cell surface through the receptor-binding domain (HA1) engaging SA moieties. Following endocytosis and trafficking to the late endosome, HA promotes fusion of the viral and endosomal membranes. The model of HA-mediated membrane fusion describes a “spring-loaded” metastable prefusion conformation at neutral pH. Dissociation of HA1 from the fusion domain (HA2) allows HA2 to undergo a cascade of conformational changes that drive membrane fusion. While extensive structural data exist for HA pre- and postfusion, and alternative conformations have been visualized and inferred, the conformational trajectory that leads to membrane fusion, including the adoption of anticipated intermediates, has never been explicitly validated. Nor has the order and timing of conformational changes and membrane fusion been determined. Here, we will utilize a multifaceted approach involving single-molecule Förster resonance energy transfer imaging, single-virion fusion, cryoelectron tomography, and molecular dynamic simulation to directly visualize the conformational trajectory undergone by HA during membrane fusion. We will explore the roles of virion morphology, HA density and cooperativity, and target membrane lipid content in mediating HA conformational changes and the mechanism of fusion. We will describe the allosteric connection between distal regions of HA that regulate the timing of fusion, drawing comparison to SARS-CoV-2 S. Finally, we will biophysically characterize the phenotypic differences between human and avian IAV strains to determine what prevents avian IAV strains from entering the human population.