Sequencing and mutational analysis of the non-coding regions of influenza A virus

The genome of influenza A virus consists of eight negative-stranded RNA segments which contain one or two coding regions flanked by the 3' and 5' non-coding regions (NCRs). Despite the importance of NCRs in replication and pathogenesis of influenza virus, sequencing of influenza virus genome has mainly been focused on coding regions of the individual genes and very limited NCR sequences are available. In this study, we sequenced the NCRs of seven influenza A virus strains of different host origin and varying pathogenicity using two recently developed methods [de Wit, E., Bestebroer, T.M., Spronken, M.I., Rimmelzwaan, G.F., Osterhaus, A.D., Fouchier, R.A., 2007. Rapid sequencing of the non-coding regions of influenza A virus. J. Virol. Methods 139, 85-89; Szymkowiak, C., Kwan, W.S., Su, Q., Toner, T.J., Shaw, A.R., Youil, R., 2003. Rapid method for the characterization of 3' and 5' UTRs of influenza viruses. J. Virol. Methods 107, 15-20]. In addition to sequence and length variation present in the segment-specific NCRs among different influenza strains, we also observed sequence variations at the fourth nucleotide of 3' NCR of polymerase genes. To evaluate the role of sequence change in the NCRs in reporter gene expression, we introduced mutations at the NCRs of two polymerase gene segments, PB1 and PA, and created the green fluorescent protein (GFP) reporter plasmids. By measuring the GFP expression level, we confirmed that single or two mutations introduced at the 3' and 5' NCRs of PB1 and PA gene could alter the protein expression levels. Our study reaffirms the importance of NCRs in influenza virus replication and further analysis of their roles will lead to better understanding of influenza pathogenesis.