Investigating the Effects of Sequence Variation and Host-Switching Mutations on the Dynamic Activation of Influenza Hemagglutinin

Abstract

The process by which influenza virions enter cells involves multiple steps that are carried out by the trimeric glycoprotein hemagglutinin (HA). In addition to recognizing receptors on the cell surface, HA mediates fusion of the viral and host cell membranes following endocytosis. The multifunctionality of HA is afforded by its ability to change conformations in response to an environmental trigger (pH). Changes in pH sensitivity and acid stability are observed across divergent subtypes of HA and due to specific mutations associated with host-switching, but it remains unclear how these differences impact HA function. Recently, the changes involved in the transition from the pre- to postfusion conformation have been characterized by techniques that probe protein dynamics, providing new mechanistic insights into the process of HA-mediated fusion. Here, we apply hydrogen/deuterium exchange mass spectrometry (HDX-MS) to study how sequence variation and host-switching mutations affect HA dynamics during acid-induced activation. Using a recombinant HA construct that we show behaves similarly to the HA ectodomain cleaved from the virus (Chapter 2), we elucidate mechanistic differences that drive changes in acid stability for H5 HA with two sets of host-switching mutations (Chapter 3). Our data indicate that host-switching mutations dampen dynamics in regions that become more exposed at low pH, including the HA1-HA2 interface and fusion peptide. This result suggests that pH-responsive regions are hotspots for adaptive changes. In Chapter 4, we show that this trend persists in light of variation in activation dynamics seen across diverse subtypes. Together these data reveal a connection between activation dynamics and acid stability that is central during adaptation to a new host.