New determinant of influenza hemagglutinin cleavability identified through random mutagenesis for acid-stabilizing mutations.

The structural instability of influenza hemagglutinin (HA) is related to its function in low pH-mediated membrane fusion, which requires prior cleavage of the premature HA0 by a host protease. Mapping the precise determinants of HA cleavability and stability has implications for risk assessment of zoonotic influenza A viruses, viral transmissibility, and vaccine production. Here, we conducted random mutagenesis on the early 2009 pandemic H1 HA, followed by selection of acid-stable viruses, and detailed profiling of the mutant HAs. We identified mutation D346N, which is located in the cleavage loop and renders H1 HA0 12-fold resistant to trypsin activation. Its cleavage by transmembrane serine protease 2 (TMPRSS2) was proved not to be affected. Likewise, we found that the poor cleavage of H16 HA0, which is unusual in carrying an N346 residue, is evident with exogenous trypsin and when non-activated particles encounter TMPRSS2 only during entry. Since H16 HA also exhibits a very low fusion pH, we propose that the gull H16N3 virus may carry a much more stable HA than other avian influenza A viruses. In addition, our random mutagenesis study on H1 HA yielded four acid-stabilizing mutations, which decrease the pH and efficiency of membrane fusion, and subtly impair the viral entry and replication in human airway-derived epithelial cells. These mutations are situated in the globular head, vestigial esterase, and membrane-proximal part of H1 HA, in regions involved in the refolding of HA at low pH. Collectively, our mutagenesis approach revealed determinants of HA cleavability and stability, with relevance for viral surveillance and vaccine production.IMPORTANCEThe presence of influenza A viruses (IAV) throughout the animal world, particularly avian species, represents a constant threat for zoonotic infections or a new influenza pandemic. To be transmissible among humans, a zoonotic IAV requires a hemagglutinin (HA) that is activated by host proteases and exhibits appropriate stability. Mapping the underlying determinants also matters for producing HA-based vaccines with high shelf stability. Through random mutagenesis and selection of acid-stable viruses, we discovered a mutation that renders HA resistant to exogenous trypsin activation. The natural occurrence of this residue in H16 HA, combined with the prominent acid stability of this HA subtype, suggests that the gull H16N3 virus may differ from other avian IAVs in carrying an environmentally stable HA. Besides, we identified four stabilizing mutations located in different parts of HA. Hence, our study delivers insight into factors that modulate HA cleavability and acid stability, with relevance for viral surveillance and vaccine production.