The Study Of The Type-specific Promoter Sequences Of Influenza A And B Viruses Affecting The Replication Mechanism Of The Viruses Between The Types

Type A and type B influenza viruses(FluA and FluB)are two major human pathogens that share common structural and functional features.FluA and FluB could reassort within each type but never between them.Although the promoters of FluA and FluB are highly conserved,there are distinct nucleotides exist.Howerver,the biological significances of the differences remain obscure.Therefore,in this study,we attempted to study the biological significances of the different promoter elements.In this study,we systematically analyzed all available promoter sequences of eight segments of FluA and FluB.We further identified that there are two representative type-specific elements.They are the 5U,6'A in FluA and the 5C,6'U in FluB.We constructed the RNP reconstitutions of FluA(A/WSN)and FluB(B/Yam),and investigated the preference of FluA and FluB polymerase for homo-or heterotypic vRNA templates and found that both of them favor homotypic templates.We further analyzed the effects of the type-specific promoter element swapping mutants on viral RNA and protein synthesis.These results indicated that the type-specific promoter element at position 5 in the 3' end of the FluA and FluB promoters are indeed involved in differentiating between FluA and FluB polymerase and also plays a critical role in regulating type-specific polymerase activities.Then we further analyzed the role of the identity of the nucleotide at position 5 in the 3' end of FluA and FluB promoters in regulating in vivo and in vitro polymerase activity,These results suggested that the identity of the nucleotide at position 5 in the 3' end of FluA and FluB promoters are important in modulating the in vivo and in vitro polymerase activities.In addition,we study the biological significance of these mutations in virus infection systems.Our results confirmed the critical role of the nucleotide at position 5 in the 3'end of FluA and FluB promoters at the virus level.Intriguingly,swapping the key element between FluA and FluB recombinant viruses caused different consequences.The replacement of the FluA-specific U5 with the FluB-specific C5 in A/WSN background could be immediately reverted to A type(U5)after 2-3 passages.In contrast,the replacement of the FluB-specific C5 with FluA-specific U5 in the B/Yam background could be maintained but with significantly reduced virus replication efficiency.This finding highlights the tolerance of nucleotide variations and the error-correcting abilities of FluA and FluB polymerases vary significantly.In conclusion,this study identified and revealed,for the first time,that the nucleotide at position 5 in the 3' end of FluA and FluB promoters is a critical type-specific determinate.Our discovery indicates that promoter variation between FluA and FluB contributes to their RNP incompatibilities,which shed new light on understanding the mechanism of the intertypic exclusion of reassortments between FluA and FluB.This work also further broad our basic knowledge on influenza type specificities and the biological significance of the influenza virus promoter.This study also revealed the transcription mechanism of FluA and FluB may be slightly varied,and the functional difference may be of great interests for future crystal structural studies.