title: 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.
abstract: 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. We found that WSN-DelNS1 viruses passaged in cells consistently adapted to gain an A14U substitution in the 3' noncoding region of the M segment of viral RNA (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 of M mRNAs. Expression of the M transcripts, M1, M2, mRNA3, and mRNA4, is regulated by alternative splicing. The A14U substitution changes the splicing donor site consensus sequence of mRNA3, altering expression of M transcripts, with M2 expression significantly increased and mRNA3 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 increases M vRNA synthesis in DelNS1 virus infection and enhances alternative splicing of M2 mRNA in the absence of other viral proteins. The findings demonstrate that NS1 is directly involved in influenza virus replication through modulation of alternative splicing of M transcripts and provide strategic information important to construction of vaccine strains with NS1 deletions.
  
  
  OBJECTIVE
  Nonstructural protein (NS1) of influenza virus has multiple functions. Besides its role in antagonizing host antiviral activity, NS1 is also believed to be involved in regulating virus replication, but mechanistic details are not clear. The NS1 protein is a virulence determinant which inhibits both innate and adaptive immunity and live attenuated viruses with NS1 deletions show promise as effective vaccines. However, deletion of NS1 causes severe attenuation of virus replication during infection, impeding functional studies and vaccine development. We characterized a replication-competent DelNS1 virus which carries an A14U substitution in the 3' noncoding region of the vRNA M segment. We found that M-A14U mutation supports virus replication through modulation of alternative splicing of mRNAs transcribed from the M segment. Our findings give insight into the role of NS1 in influenza virus replication and provide an approach for constructing replication-competent strains with NS1 deletions for use in functional and vaccine studies.
authors.affiliation: State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and Research Center of Infection and Immunology, The University of Hong Kong, Hong Kong SAR, People's Republic of China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, the University of Hong Kong, Hong Kong SAR, People's Republic of China.
authors.forename: Min
authors.initials: M
authors.lastname: Zheng
authors.affiliation: State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and Research Center of Infection and Immunology, The University of Hong Kong, Hong Kong SAR, People's Republic of China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, the University of Hong Kong, Hong Kong SAR, People's Republic of China.
authors.forename: Pui
authors.initials: P
authors.lastname: Wang
authors.affiliation: State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and Research Center of Infection and Immunology, The University of Hong Kong, Hong Kong SAR, People's Republic of China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, the University of Hong Kong, Hong Kong SAR, People's Republic of China.
authors.forename: Wenjun
authors.initials: W
authors.lastname: Song
authors.affiliation: State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and Research Center of Infection and Immunology, The University of Hong Kong, Hong Kong SAR, People's Republic of China.
authors.forename: Siu-Ying
authors.initials: SY
authors.lastname: Lau
authors.affiliation: State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and Research Center of Infection and Immunology, The University of Hong Kong, Hong Kong SAR, People's Republic of China.
authors.forename: Siwen
authors.initials: S
authors.lastname: Liu
authors.affiliation: State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and Research Center of Infection and Immunology, The University of Hong Kong, Hong Kong SAR, People's Republic of China.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Huang
authors.affiliation: State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and Research Center of Infection and Immunology, The University of Hong Kong, Hong Kong SAR, People's Republic of China.
authors.forename: Bobo Wing-Yee
authors.initials: BW
authors.lastname: Mok
authors.affiliation: State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and Research Center of Infection and Immunology, The University of Hong Kong, Hong Kong SAR, People's Republic of China.
authors.forename: Yen-Chin
authors.initials: YC
authors.lastname: Liu
authors.affiliation: National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Sciences, Xiamen University, Xiamen, People's Republic of China.
authors.forename: Yixin
authors.initials: Y
authors.lastname: Chen
authors.affiliation: State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and Research Center of Infection and Immunology, The University of Hong Kong, Hong Kong SAR, People's Republic of China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, the University of Hong Kong, Hong Kong SAR, People's Republic of China kyyuen@hku.hk hlchen@hku.hk.
authors.forename: Kwok-Yung
authors.initials: KY
authors.lastname: Yuen
authors.affiliation: State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and Research Center of Infection and Immunology, The University of Hong Kong, Hong Kong SAR, People's Republic of China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, the University of Hong Kong, Hong Kong SAR, People's Republic of China kyyuen@hku.hk hlchen@hku.hk.
authors.forename: Honglin
authors.initials: H
authors.lastname: Chen
mesh_terms: D020413:3' Untranslated Regions; D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D004285:Dogs; D016679:Genome, Viral; D057809:HEK293 Cells; D006801:Humans; D009980:Influenza A virus / Q000235:genetics* / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D008969:Molecular Sequence Data; D017354:Point Mutation; D022821:RNA Splice Sites; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000172:deficiency / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics*
chemical_list: D020413:3' Untranslated Regions; C085820:INS1 protein, influenza virus; D022821:RNA Splice Sites; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins