Influenza A Virus Hemagglutinin Is Produced in Different Disulfide-Bonded States.

Influenza A virus hemagglutinin (HA) binding to sialic acid on lung epithelial cells triggers membrane fusion and infection. Host thiol isomerases have been shown to play a role in influenza A virus infection, and we hypothesized that this role involved manipulation of disulfide bonds in HA. Analysis of HA crystal structures revealed that three of the six HA disulfides occur in high-energy conformations and four of the six bonds can exist in unformed states, suggesting that the disulfide landscape of HA is generally strained and the bonds may be labile. We measured the redox state of influenza A virus HA disulfide bonds and their susceptibility to cleavage by vascular thiol isomerases. Using differential cysteine alkylation and mass spectrometry, we show that all six HA disulfide bonds exist in unformed states in ∼1 in 10 recombinant and viral surface HA molecules. Four of the six H1 and H3 HA bonds are cleaved by the vascular thiol isomerases, thioredoxin and protein disulphide isomerase, in recombinant proteins, which correlated with surface exposure of the disulfides in crystal structures. In contrast, viral surface HA disulfide bonds are impervious to five different vascular thiol isomerases. It has been assumed that the disulfide bonds in mature HA protein are intact and inert. We show that all six HA disulfide bonds can exist in unformed states. These findings indicate that influenza A virus HA disulfides are naturally labile but not substrates for thiol isomerases when expressed on the viral surface.