The Mechanism Of ANP32 Family Protein In The Replication Of Influenza B Virus And Its Cross-species Transmission

The influenza B virus(IBV)cause seasonal influenza and unlike influenza A virus(IAV)can infect a broad range of hosts,IBV mainly infect humans and few other mammals,but fairly rare in birds.The replication mechanism and the determinants of interspecies transmission of IBV are largely unknown.Our previous study found that knockout of the ANP32 gene in mammal cells resulted in a serious decline in IBV polymerase activity,and the virus could not replicate.Moreover,the ability of avian ANP32 protein to support IBV replication was significantly lower than that of mammals,resulting in the IBV cannot replicate in avian cells.To clarify the mechanism,this study focus on the molecular mechanism of ANP32 protein supporting IBV polymerase replication and explore its effect on the post-translational modification of viral polymerase and viral RNA transport.We will identify the key sites affecting the activity of avian ANP32 protein by amino acid sequence alignment and fragment interchange of mammalian and avian ANP32 proteins,and reveal the molecular mechanism that avian ANP32 protein cannot support IBV replication.Through the study,we improved the understanding of the working principle and host adaptation mechanism of influenza B virus polymerase.In this study,we found that the host ANP32 proteins are required for the function of the IBV polymerase.Human ANP32A/B strongly supports IBV replication,while ANP32E has a limited role.Unlike human ANP32A/B,chicken ANP32A has low support activity to IBV polymerase because of a unique 33-amino-acid insert,which,in contrast,exhibits species specific support to avian influenza A virus(IAV)replication.Chicken ANP32B and ANP32E have even lower activity compared with human ANP32B/E due to specific amino acid substitutions at sites 129-130.We further revealed that the sites 129-130 affect the binding ability of ANP32B/E to IBV polymerase,while the 33-amino-acid insert of chicken ANP32A reduces its binding stability and affinity.Taken together,the features of avian ANP32 proteins limited their abilities to support IBV polymerase,which could prevent efficient replication of IBV in chicken cells.These results illustrate roles of ANP32 proteins in supporting IBV replication and may help to understand the ineffective replication of IBV in birds.Our findings reveal an important role for ANP32 proteins in IBV polymerase activity and suggest the possible molecular basis of adaptation and restriction of IBV infection in different species and provide theoretical basis for the design of antiviral drugs and the prevention and control of interspecies transmission.