Pathogenicity of the H1N1 influenza virus enhanced by functional synergy between the NP(V100I) and NA(D248N) pair

By comparing and measuring covariations of viral protein sequences from isolates of the 2009 pH1N1 influenza A virus (IAV), specific substitutions that co-occur in the NP-NA pair were identified. To investigate the effect of these co-occurring substitution pairs, the V100I substitution in NP and the D248N substitution in NA were introduced into laboratory-adapted WSN IAVs. The recombinant WSN with the covarying NP(V100I)-NA(D248N) pair exhibited enhanced pathogenicity, as characterized by increased viral production, increased death and inflammation of host cells, and high mortality in infected mice. Although direct interactions between the NP(V100I) and NA(D248N) proteins were not detected, the RNA-binding ability of NP(V100I) was increased, which was further strengthened by NA(D248N), in expression-plasmid-transfected cells. Additionally, the NA(D248N) protein was frequently recruited within lipid rafts, indirectly affecting the RNA-binding ability of NP as well as viral release. Altogether, our data indicate that the covarying NP(V100I)-NA(D248N) pair obtained from 2009 pH1N1 IAV sequence information function together to synergistically augment viral assembly and release, which may explain the observed enhanced viral pathogenicity.