Combining Mutual Information with Structural Analysis to Screen for Functionally Important Residues in Influenza Hemagglutinin

Influenza hemagglutinin mediates both cell-surface binding and cell entry by the virus. Mutations to hemagglutinin are thus critical in determining host species specificity and viral infectivity. Previous approaches have primarily considered point mutations and sequence conservation; here we develop a complementary approach using mutual information to examine concerted mutations. For hemagglutinin, several overlapping selective pressures can cause such concerted mutations, including the host immune response, ligand recognition and host specificity, and functional requirements for pH-induced activation and membrane fusion. Using sequence mutual information as a metric, we extracted clusters of concerted mutation sites and analyzed them in the context of crystallographic data. Comparison of influenza isolates from two subtypes--human H3N2 strains and human and avian H5N1 strains--yielded substantial differences in spatial localization of the clustered residues. We hypothesize that the clusters on the globular head of H3N2 hemagglutinin may relate to antibody recognition (as many protective antibodies are known to bind in that region), while the clusters in common to H3N2 and H5N1 hemagglutinin may indicate shared functional roles. We propose that these shared sites may be particularly fruitful for mutagenesis studies in understanding the infectivity of this common human pathogen. The combination of sequence mutual information and structural analysis thus helps generate novel functional hypotheses that would not be apparent via either method alone.