Inactivation mechanisms of Influenza A virus under pH conditions encountered in aerosol particles as revealed by whole-virus HDX-MS.

Multiple respiratory viruses including Influenza A virus (IAV) can be transmitted via expiratory aerosol particles, and aerosol pH was recently identified as a major factor influencing airborne virus infectivity. For indoor air, small exhaled aerosols undergo rapid acidification to pH [~]4. IAV is known to be sensitive to mildly acidic conditions encountered within host endosomes, however, it is unknown whether the same mechanisms could mediate viral inactivation within the more acidic aerosol micro-environment. Here, we identified that transient exposure to pH 4 caused IAV inactivation by a two-stage process, with an initial sharp decline in infectious titers that was mainly attributed to premature attainment of the post-fusion conformation of viral protein haemagglutinin (HA). Changes to HA were observed by hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) as early as 10 seconds post-exposure to acidic conditions. In addition, virion integrity was partially but irreversibly affected by acidic conditions. This was attributed to a progressive unfolding of the internal matrix protein 1 (M1), and aligned with a more gradual decline in viral infectivity with time. In contrast, no acid-mediated changes to the genome or lipid envelope were detected. Our HDX-MS data are in agreement with other more labor-intensive structural analysis techniques such as X-ray crystallography, highlighting the usefulness of whole-virus HDX-MS for multiplexed protein analyses, even within enveloped viruses such as IAV. Improved understanding of respiratory virus fate within exhaled aerosols constitutes a global public health priority, and information gained here could aid development of novel strategies to control the airborne persistence of seasonal and/or pandemic influenza in the future.