Pattern of nucleotide substitution in the overlapping nonstructural genes of influenza A virus and implication for the genetic diversity of the H5N1 subtype

In viruses under strong pressure to minimize genome size, overlapping genes represent a fine strategy to condense a maximum amount of information into short nucleotide sequences. Here, we investigated the evolution of the genes encoding the nonstructural proteins NS1 and NS2 of influenza A virus (IAV), which are one of the best characterized cases of gene overlap. By a detailed analysis of about four hundred sequences grouped into 11 IAV subtypes, we found that the overlapping coding region of the NS1 gene shows a significant increase of the rate of nonsynonymous change, with respect to its nonoverlapping counterpart. The same feature was observed in the overlapping coding region of the NS2 gene. Such a variation pattern, which implies the occurrence of several amino acid substitutions in the protein regions encoded by overlapping frames, is different from the pattern of constrained evolution typical of other viral overlapping-gene systems. Amino acid sequence analysis of the NS1 and NS2 proteins revealed that some nonsynonymous substitutions, located in the region of gene overlap, play a critical role in shaping the genetic diversity of the highly pathogenic subtype H5N1. Since both proteins contribute to disease pathogenesis by affecting many virus and host-cell processes, information provided by this study should be useful to highlight the impact of nonstructural gene variation on the pathogenicity of H5N1 viruses.