Random mutagenesis of influenza hemagglutinin identifies new sites which modulate its acid-stability and cleavability

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. The precise determinants underlying the stability and cleavability of HA remain to be fully understood and have implications for risk assessment of zoonotic influenza A viruses (IAV), viral transmissibility and vaccine production. To address this, we conducted random mutagenesis on early 2009 pandemic H1 HA, followed by selection of acid-stable viruses and detailed profiling of the mutant HAs. This resulted in identification of four mutations, which increase the acid-stability and decrease the fusion-promoting activity of H1 HA, without compromising viral entry and replication in cells. The newly recognized 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. A fifth mutation, D346N, is located in the cleavage loop and renders H1 HA0 12-fold resistant to trypsin activation, whereas its cleavage by transmembrane serine protease 2 (TMPRSS2) is not affected. Along this line, we found that the poor cleavage of H16 HA0, which is unusual in carrying an N346 residue, only applies when it is performed by extracellular proteases. Since H16 HA also exhibits a very low fusion pH, we propose that gull H16N3 virus may carry a much more stable HA than other avian IAVs. Collectively, our mutagenesis approach revealed new determinants of HA stability and cleavability, 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 mutations in the viral hemagglutinin (HA) that increase the acid-stability of this mediator of viral entry. Understanding the determinants of HA stability is also important to produce vaccines with high shelf-stability. By combining random mutagenesis with selection of acid-stable viruses, we identified new stabilizing mutations located in different parts of HA. Besides, we discovered a mutation that renders HA resistant to cleavage by extracellular proteases. Since this residue is naturally occurring in H16 HA, we propose that the gull H16N3 virus may differ from other avian IAVs in carrying an environmentally stable HA. Hence, our study delivers new insight in factors that modulate HA acid-stability and cleavability, with relevance for viral surveillance and vaccine production.