Study On The Roles Of HnRNPs In Influenza A Virus Infected Cells

Influenza A virus is the main pathogen to cause flu,which seriously threatens human life and health.The genome of influenza A virus is single-strand and negative-sense,which consist of eight segments.The new strains of influenza A virus with different genome can emerge for the mutation and genome reassortment.Therefore its important to explore the mechanism of the competition between influenza A virus and host cell.After enter into the cells,the membranes of influenza A virus and endosomal fuse and the viral ribonucleoprotein(v RNP)is released into the cytoplasm and transported into nucleus.The transcription and genome replication of influenza A virus happen in nucleus which is rare for RNA virus.The hn RNPs is a most abundant RNA binding protein family which interacts with pre-mRNA to form the hn RNP perticles and participates the modification,stabilization,nuclear export and translation of mRNA.A few kinds of hn RNPs have been reported to function during influenza A virus infection.However,it is still unclear how do other kinds of the hn RNPs work during influenza A virus infection.To explore whether the hn RNPs are regulated when cells are infected with influenza A virus,we examined the mRNA and protein level at different time post infection.Interestingly,different kinds of hn RNPs tested(hn RNP AB,hn RNP A1,hn RNP A2B1,hn RNP F,hn RNP K and hn RNP U)were all up-regulated when cells were infected with influenza A virus.Furthermore,we discovered that the NS1 and NP of H7N9 influenza A virus both showed localization with hn RNP AB,hn RNP A1 and hn RNP U instead of hn RNP A2B1.Meanwhile,we observed that the hn RNP A2B1 and hn RNP U could change the cellular localization of NS1 and NP which means that the hn RNPs may change the function of NS1 and NP.In another way,we found that the NS1 instead of NP protein inhibit the interaction between hn RNP C2 and hn RNP A1 or hn RNP AB,which indicated that the NS1 protein of influenza A virus may destroy the accumulation of hn RNPs.By immunoprecipitation we found that hn RNP AB and hn RNP U could interact with kinds of viral proteins,which suggested that hn RNP AB and hn RNP U play critical roles during influenza A virus infection.To investigate the effect of hn RNPs on the replication of influenza A virus,we knocked down the hn RNP AB,hn RNP A1 or hn RNP U and the cells were then infected with influenza A virus.The results showed that the replication of influenza A virus was up-regulated when the hn RNP AB,hn RNP A1 or hn RNP U was knocked down.Collectively,hn RNPs are important for regulating the life cycle of IAV and further study is critical for developing a new antivirus strategy.Heterogeneous nuclear ribonucleoproteins AB(hnRNP AB)is a member of hn RNPs which play critical roles in RNA splicing,nuclear export and translation.However,the role of hn RNP AB in cell life cycle is still poorly understood.In this study,we prove that the hn RNP AB acts as a mRNA nuclear retention factor to inhibit the replication of influenza A virus.By further study,we found that hn RNP AB weakens the ALY-mRNA binding by interfering the interaction between UAP56 and ALY.Furthermore,we discovered that hn RNP AB inhibits the NXF1-mRNA binding significantly by directly binding to the RNA binding domain of NXF1.Interestingly,the influenza A virus NP binds to hn RNP AB which is mediated by RNA and enhances the nuclear retention of viral mRNA to inhibit virus replication.We found that NP cooperates with hn RNP AB to inhibit the interaction of UAP56 and ALY significantly which lead to the significant down-regulation of ALY-mRNA binding.For the reason that hnRNPs are important factors for cellular pre-mRNA splicing,it is necessary to investigate the impact of influenza A virus on cellular pre-mRNA splicing.In this study,we proved that the splicing of pre-mRNA of RIG-I and IRF3 were all interfered.The 2,3 intron of RIG-I was mis-spliced and the splicing of 4,5intron of IRF3 were inhibited when cells were infected with influenza A virus which showed two different ways for influenza A virus to regulate the cellular splicing.Further study proved that the NS1 protein of influenza A virus is the key factor leading to the mis-splicing of RIG-I pre-mRNA.For the inhibition of splicing of IRF3pre-mRNA,we proved that the infection of influenza A virus promoted the binding of hn RNP U to 4,5 intron of IRF3 pre-mRNA which leads to the inhibition of the splicing.Moreover,the infection of influenza A virus leads to the up-regulation and the change of the localization in nucleus of sn RNA U6.Meanwhile,nuclear speckle,the storage structure of the component for splicing,is affected by influenza virus infection with its size decreasing and its number increasing.Meanwhile,we demonstrated that the R19 residue of NS1 is critical for its RNA binding and nuclear localization.Furthermore,the residue R19 of NS1 was identified to be critical for regulating M1 mRNA splicing and NS1 nuclear export.