Comparative Analysis of Mutational Constraints Across Influenza Hemagglutinin Subtypes

Abstract

H7 avian influenza represents a significant pandemic threat due to its ability to cause severe disease in humans and its potential for cross-species transmission. To enhance our understanding of this virus and improve pandemic preparedness, we developed a comprehensive deep mutational scanning system to systematically assess the functional effects of mutations in H7 hemagglutinin (HA). This system utilizes a pseudotyped lentiviral platform to precisely determine how individual mutations affect critical viral functions including HA-mediated cell entry, pH stability, and receptor binding specificity. Through this approach, we generated new insights into H7 evolutionary constraints and identified novel entry mechanisms that challenge traditional models of influenza host range determination. The experimental framework we established provides a safe and generalizable method for mapping the functional landscape of viral entry proteins, offering valuable tools for assessing pandemic potential across diverse influenza subtypes and other emerging viruses. In chapter 2, we examine the development of an improved pH stability assay capable of measuring how mutations affect HA protein stability under acidic conditions. This enhanced methodology addresses key limitations of traditional approaches by incorporating virus reconcentration steps, enabling high-throughput analysis of HA variants with varying pH activation thresholds. We demonstrate the assay's utility using H5N1 hemagglutinin, successfully identifying both stabilizing and destabilizing mutations that influence viral environmental persistence and transmission potential. In chapter 3, we present the first comprehensive deep mutational scanning analysis of H7 hemagglutinin, systematically measuring the functional effects of nearly every possible amino acid mutation on viral cell entry. Our analysis reveals that H7 can efficiently bind both human-type α2-6 and avian-type α2-3 linked sialic acid receptors, confirming its dual receptor capability. We identify critical functional constraints across different HA domains and demonstrate how specific mutations present in circulating H7N9 strains enhance human receptor binding, providing insights into the virus's pandemic adaptation potential. In chapter 4, we examine how mutational constraints differ among influenza HA subtypes by comparing deep mutational scanning datasets from H3, H5, and H7. Despite sharing highly conserved protein structures and identical functional mechanisms, these HA subtypes display dramatically different tolerance patterns for specific mutations. Our analysis reveals that approximately half of all HA sites show significantly diverged amino acid preferences across subtypes, demonstrating how evolutionary sequence changes can rewire protein constraints even when overall structure and function remain preserved. Finally, in chapter 5, we investigate an alternative entry mechanism through which H7 can utilize Major Histocompatibility Complex class II (MHCII) molecules as cellular receptors. This discovery reveals that influenza entry pathways extend beyond traditional sialic acid-dependent mechanisms and may provide previously unrecognized routes for cross-species transmission. Through systematic mutational analysis, we identify putative MHCII binding interfaces and demonstrate that this alternative entry pathway operates through molecular determinants distinct from canonical receptor binding sites, with important implications for viral host range and pandemic risk assessment.