Hemagglutinin Stability Determines Influenza A Virus Susceptibility to a Broad-Spectrum Fusion Inhibitor Arbidol

Understanding
mechanisms of resistance to antiviral inhibitors
can reveal nuanced features of targeted viral mechanisms and, in turn,
lead to improved strategies for inhibitor design. Arbidol is a broad-spectrum
antiviral that binds to and prevents the fusion-associated conformational
changes in the trimeric influenza A virus (IAV) hemagglutinin (HA).
The rate-limiting step during the HA-mediated membrane fusion is the
release of the hydrophobic fusion peptides from a conserved pocket
on HA. Here, we investigated how destabilizing or stabilizing mutations
in or near the fusion peptide affect viral sensitivity to Arbidol.
The degree of sensitivity was proportional to the extent of fusion-peptide
stability on the prefusion HA: stabilized mutants were more sensitive,
and destabilized ones were resistant to Arbidol. Single-virion membrane
fusion experiments for representative wild-type (WT) and mutant viruses
demonstrated that resistance is a direct consequence of fusion-peptide
destabilization not requiring reduced Arbidol binding to HA. Our results
support the model whereby the probability of individual HAs extending
to engage the target membrane is determined by the composite of two
critical forces: a “tug” on the fusion peptide by HA
rearrangements near the Arbidol binding site and the key interactions
stabilizing the fusion peptide in the prefusion pocket. Arbidol increases
and destabilizing mutations decrease the free-energy cost for fusion-peptide
release, accounting for the observed resistance. Our findings have
broad implications for fusion inhibitor design, viral mechanisms of
resistance, and our basic understanding of HA-mediated membrane fusion.