A Single Amino Acid in the Stalk Region of the H1N1pdm Influenza Virus HA Protein Affects Viral Fusion, Stability and Infectivity

The 2009 H1N1 pandemic (H1N1pdm) viruses have evolved to contain an E47K substitution in the HA2 subunit of the stalk region of the hemagglutinin (HA) protein. The biological significance of this single amino acid change was investigated by comparing A/California/7/2009 (HA2-E47) with a later strain, A/Brisbane/10/2010 (HA2-K47). The E47K change was found to reduce the threshold pH for membrane fusion from 5.4 to 5.0. An inter-monomer salt bridge between K47 in HA2 and E21 in HA1, a neighboring highly conserved residue, which stabilized the trimer structure, was found to be responsible for the reduced threshold pH for fusion. The higher structural and acid stability of the HA trimer caused by the E47K change also conferred higher viral thermal stability and infectivity in ferrets, suggesting a fitness advantage for the E47K evolutionary change in humans. Our study indicated that the pH of HA fusion activation is an important factor for influenza virus replication and host adaptation. The identification of this genetic signature in the HA stalk region that influences vaccine virus thermal stability also has significant implications for influenza vaccine production. Author Summary Influenza viruses cause seasonal epidemics and occasional pandemics, representing a threat to public health. The trimeric hemagglutinin (HA) surface glycoprotein mediates viral entry and plays important roles in viral host restriction, transmission and pathogenesis. The HA protein binds to the receptor on the cell via the head region, and mediates the low pH-triggered viral and cellular membrane fusion via the stalk region. The 2009 H1N1 pandemic (H1N1pm) viruses have been circulating in humans since 2009. A single amino acid change was found in the stalk region of recent H1N1pdm strains. In this study, we revealed that this amino acid change, by stabilizing the trimer structure, lowered the pH for fusion and exhibited a higher acid stability. Accordingly, the H1N1pdm with this change showed higher thermal stability after high temperature treatment. Furthermore, the H1N1pdm with this change had higher infectivity in ferrets, suggesting that recent H1N1pdm viruses have evolved to be more adapted in humans. Our data not only indicate the importance of the fusion activation pH to viral replication and host adaptation, but also provide solutions to improve the shelf-life of live vaccines in vaccine manufacture.