The Molecular Mechanism Of The Interaction Between Avian Host Protein HnRNPAB And PB2 Affecting The Replication Of Avian Influenza Virus

When AIV attaches the cells,the virus particles enter the cells and rely on much more host factors to complete its own transcription and replication and other biological processes.PB2 subunit is one of the virus particles of AIV,which acts as a pivotal role in the virus genomes transcription and replication of AIV.There are many host proteins that could associate with the PB2 protein of influenza A virus in host cells.Moreover,these host proteins are mainly involved in the nuclear localization of viral proteins,transcription and replication of viral genomes and so on.Although there exist many host factors which associate with the viral PB2 protein,the functions of many interacting proteins and their effects on virus replication remain unclear.The heterogeneous nuclear ribonucleoprotein family(hnRNPs)are important for the process of cell biology and virus replication.Our past studies have found that human-derived hnRNPAB could inhibit the polymerase activity and virus replication of mammalian adaptive virus strains.And silencing of hnRNPAB found that the polymerase activity and virus replication were enhanced,which further confirmed the importance of hnRNPAB in the biological process of IAV.In order to explore the importance of avian-derived hnRNPAB in the process of AIV infection,we carried out this study.The protein coding regions of PB2 protein of AIV were cloned into the p CAGGS-myc expression vector and the coding regions of chicken hnRNPAB were cloned into the p CAGGS-flag expression vector at first.DF-1 cells were transfected with the eukaryotic expression vectors of hnRNPAB and PB2 for 24 hours.Confocal microscope results showed that there was a co-localization phenomenon between hnRNPAB and PB2 protein and these two proteins were mainly colocalizated with each other in the nucleus.Subsequently,Co-Immunoprecipitation experiment was used to further verify whether there was an interaction between hnRNPAB and PB2 protein.We found that PB2 protein could only be displayed at the presence of hnRNPAB,indicating that there was an association between the PB2 protein and the host protein hnRNPAB.hnRNPAB also belongs to RNA binding protein with RNA binding ability.Therefore,RNA immunoprecipitation experiment found that PB2 m RNA could only be immunoprecipitated by flag antibody and then could be amplified by RT-PCR at the presence of flag-hnRNPAB,indicating that hnRNPAB could bind to PB2 m RNA.In conclusion,hnRNPAB bound with PB2 protein and m RNA of AIV.Secondly,based on the discovery of the interaction between hnRNPAB and PB2 and m RNA,this study continued to explore the effect of hnRNPAB on the protein expression and m RNA levels of PB2 and ribonucleoprotein complex.DF-1 cells were co-transfected with hnRNPAB and PB2 eukaryotic expression vectors.After 24 hours post-transfection,we found that PB2 protein expression of AIV was inhibited by overexpression of hnRNPAB via Western blot,and results also showed that overexpression of hnRNPAB had no effect on PB2 m RNA by q RT-PCR,indicating that although hnRNPAB could bind to PB2 m RNA,it did not affect the stability of PB2 m RNA to further decrease PB2 protein expression.Furthermore,the effect of hnRNPAB on PB2 protein expression was verified by virus infection test.The results demonstrated that overexpression of hnRNPAB inhibited the expression of PB2 protein after 24 hours post-infection,and the expression of PB2 protein increased when endogenous hnRNPAB protein was silenced by si RNA targeting hnRNPAB,indicating that hnRNPAB was a negative factor of PB2 protein expression.Then we continued to explore the effect of hnRNPAB on the nucleocytoplasmic distribution of PB2 m RNA.The nucleocytoplasmic separation experiment found that hnRNPAB inhibited the nuclear export of PB2 m RNA,indicating that hnRNPAB could regulate the nucleocytoplasmic distribution of PB2 m RNA and affect the expression level of PB2 protein.Each virionRNA segment is associated with RNA-dependent RNA polymerase complex(PB1,PB2 and PA subunit)and bound with NP to assemble the virus ribonucleoprotein(v RNP)complex that played the core status in viral replication and transcription.In this study,we also found the expression of PA,PB1 and NP proteins were decreased by overexpression of hnRNPAB,but the m RNA levels of PA,PB1 and NP were not changed,which indicated that hnRNPAB had a significant effect on the protein levels of v RNPs.We further explored the effect of hnRNPAB on polymerase activity and virus replication of AIV.Polymerase activity assays showed that hnRNPAB had significant inhibitory effect on the polymerase activity of PR8 PB2 K627 E and H9N2 strains.We also found that hnRNPAB inhibited the expression of NP protein and virus titers of H9N2.si RNA experiment results found that when expression of endogenous hnRNPAB was decreased,the expression of NP protein and virus titers were increased,indicating that hnRNPAB was a negative regulatory factor in the replication of AIV.Overexpression of hnRNPAB in DF-1 and 293 T cells and infected with mammalian adaptive PR8 strains showed that hnRNPAB also inhibited the polymerase activity,but did not affect the replication level of PR8 strain,which indicated the difference of hnRNPAB on AIV and mammalian adaptive strains.hnRNPAB could affect the replication of AIV,and during the process of virus infection,the protein expression levels and nucleocytoplasmic distribution of some members of hnRNPs family would be affected.Therefore,we used the nuclear separation experiment to explore the effect of AIV infection on endogenous hnRNPAB protein.We found that virus infection had no influence on endogenous hnRNPAB protein expression in DF-1 cells,but it resulted in the nuclear retention of hnRNPAB protein,which indicated that AIV infection might enhance the nuclear enrichment of hnRNPAB to inhibit the replication of AIV.In addition,results also showed that hnRNPAB inhibited the replication ability of H7N1 virus after 24 hours post-infection,further confirming the inhibition of hnRNPAB on AIV.In conclusion,overexpression of host factor hnRNPAB could reduce the nuclear output of PB2 m RNA and inhibit the protein level of PB2,which could further negatively regulate the polymerase activity and the replication of AIV.We also found that AIV infection caused the nuclear retention of endogenous hnRNPAB,which played an active role in the process of decreasing the replication of AIV in the nucleus as an intranuclear protein.These results would provide useful information for understanding the specific mechanism of hnRNPs family in the process of AIV infection.