Local structural changes of the influenza A virus ribonucleoprotein complex by single mutations in the specific residues involved in efficient genome packaging

The influenza A virus genome consists of eight single-stranded negative-sense RNA segments. The noncoding regions located at the 3′- and 5′- ends of each segment are necessary for genome packaging, and the terminal coding regions are required to precisely bundle the eight segments. However, the nucleotide residues important for genome bundling are not defined. Here, we introduced premature termination codons in the hemagglutinin (HA) or matrix protein 2 (M2) gene and constructed virus libraries containing random sequences in the terminal coding regions. Using these virus libraries, we identified nucleotide residues involved in efficient virus propagation. Viral genome packaging was impaired in viruses that contained single mutations at these identified residues. Furthermore, these single mutations altered the local structure of the viral ribonucleoprotein complex. Our results show that specific nucleotide residues in the viral protein coding region are involved in forming the precise structure of the viral ribonucleoprotein complex. •To analyze the role of bundling signals, we constructed a new system.•Nucleotide residues involved in influenza virus propagation are identified.•Viral genome packaging was impaired in mutant viruses.•The local structure of vRNP was altered by introducing these single mutations.