A soluble domain of the membrane-anchoring chain of influenza virus hemagglutinin (HA(2)) folds in Escherichia coli into the low-pH-induced conformation

The extensive refolding of the membrane-anchoring chain of hemagglutinin (HA) of influenza virus (termed HA(2)) in cellular endosomes, which initiates viral entry by membrane fusion, suggests that viral HA is metastable. HA(2) polypeptide residues 38-175 expressed in Escherichia coli are reported here to fold in vivo into a soluble trimer. The structure appears to be the same as the low-pH-induced conformation of viral HA(2) by alpha-helical content, thermodynamic stability, protease dissection, electron microscopy, and antibody binding. These results provide evidence that the structure of the low-pH-induced fold of viral HA(2) (TBHA(2)) observed crystallographically is the lowest-energy-state fold of the HA(2) polypeptide. They indicate that the HA(2) conformation in viral HA before low pH activation of its fusion potential is metastable and suggest that removal of the receptor-binding chain (HA(1)) is enough to allow HA(2) to adopt the stable state. Further, they provide direct evidence that low pH is not required to form the membrane-fusion conformation but acts to make this state kinetically accessible in viral HA.