Effect Of PB2 Protein 576E On H3N2 Canine Influenza Virus Mammalian Adaptability And Its Mechanism

H3N2 Canine influenza virus(CIV)is an infectious respiratory virus transmitted from avian influenza virus to dogs across the host barrier and can quickly spread among dogs.It is widely prevalent in many Asian countries and the United States,and is commonly accompanied with secondary infections,causing great harm to dog health.There were two strains with different replicabilities in mice isolated in the our previous research: a strain of avian-origin H3N2 CIV(A/canine/Zhejiang/01/2012)(designated ZJ01),which replicated well in mice,but could not infect poultry;The other strain of H3N2 avian influenza virus A/chicken/Shanghai/D6/2012(designated SHD6),hardly replicated in mice.When the PB2 gene of the ZJ01 was replaced by SHD6,the ZJ01 virus could not be successfully rescued.In contrast,ZJ01-PB2 significantly enhanced SHD6 virus to replicate well in mice.This study focused on clarifying the adaptability and mechanism of PB2 gene for ZJ01 virus in mammals.Polymerase activity assays were performed to detect polymerase activity with one polymerase gene-replaced or double-genes substitutions.The result demonstrated that PB2 gene played a major role in the polymerase activity of ZJ01 virus.We detected the polymerase activities after replacing the five functional regions of the PB2 protein.The result indicated that 627-domain significantly enhanced the polymerase activity of ZJ01.There were four different amino acid residues in 627-domain between ZJ01 and SHD6.The polymerase activity was measured by single amino acid substitution,the results demonstrated that 576 E amino acid site in the PB2 protein significantly increased the polymerase activity of both ZJ01 and SHD6,suggesting that the 576 E at 627-domain was a key amino acid site for regulating the polymerase activity of ZJ01 virus.To determine whether the PB2-576 E affects the replication of ZJ01 virus,we rescued rZJ01?r ZJ01-PB2-E576 K,rSHD6 and rSHD6-PB2-K576 E by using reverse genetics and compared their replication capabilities with each parental virus in mice.The result demonstrated that the viruses containing PB2-576E(rZJ01 and rSHD6-PB2-K576E)replicated better in mice than viruses containing PB2-E576 K in the same background.Meanwhile,we also detected the growth curves of rZJ01?rZJ01-PB2-E576 K,rSHD6 and rSHD6-PB2-K576 E in different host cells(MDCK or CEFs).The results show that on MDCK,PB2-576 E can significantly improve the replication ability of r-ZJ01 or r-SHD6,but on CEF,PB2-576E/ K has no obvious effect on replication of r-ZJ01 or r-SHD6.The result indicated that PB2-576 E is a key amino acid site that affects the replication of ZJ01 virus on MDCK in mammalian cells.ANP32A has been recently identified as a cellular co-factor affecting polymerase activity of influenza viruses by interacting with 627-domain of PB2.Therefore,we investigated whether the PB2-576 E interacts with the host protein ANP32 A.By co-immunoprecipitation assay,we found that PB2-576 E has significantly stronger interaction with different hosts ANP32 A than that of PB2-576 K.In a further analysis,different species ANP32 A were overexpressed in 293 T cells and their effects on the polymerase activities were detected.The result demonstrated that PB2-E576 K significantly reduced the polymerase activity of ZJ01,and overexpression of human or canine ANP32 A could not increase the polymerase activities of ZJ01-PB2-E576 K and SHD6.These results suggested that mammalian ANP32 A can only recognize PB2-576 E,which improved the polymerase activity of r-ZJ01 higher than that of rZJ01-PB2-E576 K.It suggested that the interaction between PB2-576 E and mammalian ANP32 A is a necessary condition for up-regulating the polymerase activity of ZJ01.Based on the above results,PB2-576 E regulates the polymerase activity of ZJ01 virus in mammals by enhancing the interaction with mammalian host protein ANP32 A,so that the virus has a strong replication ability in mammals,which lay the foundation for a deeply understanding of the replication and adaptation mechanisms of H3N2 CIV in mammals.