title: The choice of alternative 5' splice sites in influenza virus M1 mRNA is regulated by the viral polymerase complex.
abstract: The influenza virus M1 mRNA has two alternative 5' splice sites: a distal 5' splice site producing mRNA3 that has the coding potential for 9 amino acids and a proximal 5' splice site producing M2 mRNA encoding the essential M2 ion-channel protein. Only mRNA3 was made in uninfected cells transfected with DNA expressing M1 mRNA. Similarly, using nuclear extracts from uninfected cells, in vitro splicing of M1 mRNA yielded only mRNA3. Only when the mRNA3 5' splice site was inactivated by mutation was M2 mRNA made in uninfected cells and in uninfected cell extracts. In influenza virus-infected cells, M2 mRNA was made, but only after a delay, suggesting that newly synthesized viral gene product(s) were needed to activate the M2 5' splice site. We present strong evidence that these gene products are the complex of the three polymerase proteins, the same complex that functions in the transcription and replication of the viral genome. Gel shift experiments showed that the viral polymerase complex bound to the 5' end of the viral M1 mRNA in a sequence-specific and cap-dependent manner. During in vitro splicing catalyzed by uninfected cell extracts, the binding of the viral polymerase complex blocked the mRNA3 5' splice site, resulting in the switch to the M2 mRNA 5' splice site and the production of M2 mRNA.
authors.affiliation: Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08855-1179, USA.
authors.forename: S R
authors.initials: SR
authors.lastname: Shih
authors.forename: M E
authors.initials: ME
authors.lastname: Nemeroff
authors.forename: R M
authors.initials: RM
authors.lastname: Krug
mesh_terms: D017398:Alternative Splicing; D001483:Base Sequence; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009980:Influenza A virus / Q000378:metabolism*; D007473:Ion Channels / Q000096:biosynthesis*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012322:RNA Precursors / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis; D017381:Spliceosomes / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014763:Viral Matrix Proteins / Q000096:biosynthesis*; D000086082:Viroporin Proteins
chemical_list: D012321:DNA-Directed RNA Polymerases; D007473:Ion Channels; D012322:RNA Precursors; D012333:RNA, Messenger; D012367:RNA, Viral; D014763:Viral Matrix Proteins; C031540:M-protein, influenza virus; C488936:M1 protein, Influenza A virus; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins