Functional significance of the hemadsorption activity of influenza virus neuraminidase and its alteration in pandemic viruses

Human influenza viruses derive their genes from avian viruses. The neuraminidase (NA) of the avian viruses has, in addition to the catalytic site, a separate sialic acid binding site (hemadsorption site) that is not present in human viruses. The biological significance of the NA hemadsorption activity in avian influenza viruses remained elusive. A sequence database analysis revealed that the NAs of the majority of human H2N2 viruses isolated during the influenza pandemic of 1957 differ from their putative avian precursor by amino acid substitutions in the hemadsorption site. We found that the NA of a representative pandemic virus A/Singapore/1/57 (H2N2) lacks hemadsorption activity and that a single reversion to the avian-virus-like sequence (N367S) restores hemadsorption. Using this hemadsorption-positive NA, we generated three NA variants with substitutions S370L, N400S and W403R that have been found in the hemadsorption site of human H2N2 viruses. Each substitution abolished hemadsorption activity. Although, there was no correlation between hemadsorption activity of the NA variants and their enzymatic activity with respect to monovalent substrates, all four hemadsorption-negative NAs desialylated macromolecular substrates significantly slower than did the hemadsorption-positive counterpart. The NA of the 1918 pandemic virus A/Brevig Mission/1/18 (H1N1) also differed from avian N1 NAs by reduced hemadsorption activity and less efficient hydrolysis of macromolecular substrates. Our data indicate that the hemadsorption site serves to enhance the catalytic efficiency of NA and they suggest that, in addition to changes in the receptor-binding specificity of the hemagglutinin, alterations of the NA are needed for the emergence of pandemic influenza viruses

Prevalence of Epistasis in the Evolution of Influenza A Surface Proteins

The surface proteins of human influenza A viruses experience positive selection to escape both human immunity and, more recently, antiviral drug treatments. In bacteria and viruses, immune-escape and drug-resistant phenotypes often appear through a combination of several mutations that have epistatic effects on pathogen fitness. However, the extent and structure of epistasis in influenza viral proteins have not been systematically investigated. Here, we develop a novel statistical method to detect positive epistasis between pairs of sites in a protein, based on the observed temporal patterns of sequence evolution. The method rests on the simple idea that a substitution at one site should rapidly follow a substitution at another site if the sites are positively epistatic. We apply this method to the surface proteins hemagglutinin and neuraminidase of influenza A virus subtypes H3N2 and H1N1. Compared to a non-epistatic null distribution, we detect substantial amounts of epistasis and determine the identities of putatively epistatic pairs of sites. In particular, using sequence data alone, our method identifies epistatic interactions between specific sites in neuraminidase that have recently been demonstrated, in vitro, to confer resistance to the drug oseltamivir; these epistatic interactions are responsible for widespread drug resistance among H1N1 viruses circulating today. This experimental validation demonstrates the predictive power of our method to identify epistatic sites of importance for viral adaptation and public health. We conclude that epistasis plays a large role in shaping the molecular evolution of influenza viruses. In particular, sites with , which would normally not be identified as positively selected, can facilitate viral adaptation through epistatic interactions with their partner sites. The knowledge of specific interactions among sites in influenza proteins may help us to predict the course of antigenic evolution and, consequently, to select more appropriate vaccines and drugs