An A14U substitution in the 3' noncoding region of the M segment of viral RNA supports replication of influenza virus with an NS1 deletion by modulating alternative splicing of M segment mRNAs

The NS1 protein of influenza virus has multiple functions and is a determinant of virulence. Influenza viruses with NS1 deletions (DelNS1 influenza viruses) are a useful tool for studying virus replication and can serve as effective live attenuated vaccines, but deletion of NS1 severely diminishes virus replication, hampering functional studies and vaccine production.Wefound that WSN-DelNS1 viruses passaged in cells consistently adapted to gain an A14U substitution in the 3' noncoding region of theMsegment of viralRNA (vRNA) which restored replicative ability. DelNS1-M-A14U viruses cannot inhibit interferon expression in virus infected-cells, providing an essential model for studying virus replication in the absence of the NS1 protein. Characterization of DelNS1-M-A14U virus showed that the lack of NS1 has no apparent effect on expression of other viral proteins, with the exception ofMmRNAs. Expression of theMtranscripts, M1, M2, mRNA3, and mRNA4, is regulated by alternative splicing. The A14U substitution changes the splicing donor site consensus sequence of mRNA3, altering expression ofMtranscripts, withM2expression significantly increased andmRNA3 markedly suppressed in DelNS1-M-A14U, but not DelNS1-M-WT, virus-infected cells. Further analysis revealed that the A14U substitution also affects promoter function during replication of the viral genome. The M-A14U mutation increasesMvRNA synthesis in DelNS1 virus infection and enhances alternative splicing ofM2mRNAin the absence of other viral proteins. The findings demonstrate that NS1 is directly involved in influenza virus replication through modulation of alternative splicing ofMtranscripts and provide strategic information important to construction of vaccine strains with NS1 deletions.