HnRNPM Regulates Influenza A Virus Replication And Affects Interspecies Transmission

Influenza A virus is an important human viral pathogen.It causes occasional pandemics and annual epidemics,seriously threatening human health and life worldwide.When zoonotic influenza virus with novel antigenicity acquires the ability to across species from birds to humans,it most likely causes influenza pandemics.Host range determinates of influenza A virus are based on virus-host specific interactions.Understanding the mechanism of species-specific virus-host interactions is a key aspect in the research of interspecies transmission of influenza A viruses.Host range determinates include receptor preference,vRNP transport,and viral polymerase activities and evasion of the host antiviral response.These processes require specific interplay between host factors and viral components.We carried out an initial proteomic analysis to screen for vRNPs(WSN)-associated host proteins.16 preliminary proteins in total were identified as vRNP interacting proteins,including hnRNPM,RPLPO,and the key host-determinate host factor ANP32A.We subsequently verified that human hnRNPM regulates influenza virus replication by interacting with viral vRNPs.The heterogeneous nuclear ribonucleoprotein M(hnRNPM)belongs to the family of heterogeneous nuclear ribonucleoproteins,a family of nucleocytoplasmic shuttling RNA-binding proteins that were originally identified based on their association with pre-mRNAs.It has been reported that hnRNPM is involved in pre-mRNA splicing and diverse aspects of RNA metabolism,including translational control,telomere biogenesis,mRNA stability,and trafficking.Interestingly,alignment of the homologous hnRNPMs from human and avian,we found that human hnRNPMs(Hu-hnRNPM)contains additional 34 amino acids in comparison with avian hnRNPMs(Av-hnRNPM).We found that the Hu-hnRNPM could positively regulates both human and avian influenza virus replication,while the Av-hnRNPM only positively regulates avian influenza virus replication.To study the mechanism(s)by which hnRNPMs regulate influenza A virus replication,we first confirmed the role of the hnRNPMs in viral RNA synthesis in infected human-derived A549 cells.Primer extension analysis revealed that Hu-hnRNPM and Av-hnRNPM could differentially regulate the RNA synthesis of influenza A virus in human-and chicken-derived cells.We further confirmed that Hu-hnRNPM facilitates viral primary transcription while Av-hnRNPM does not in human A549 cells.This differential regulation of viral RNA synthesis were further confirmed in a mini-replicon system and in in in vitro transcription systems.Interestingly,in consistent with viral infection regulations,we found that the 34 amino acids in Hu-hnRNPMs is the key site responsible for upregulating human and avian influenza virus RNA synthesis while the Av-hnRNPM only supports for avian influenza virus RNA synthesis.We further examined the associations between cellular hnRNPM and viral vRNP by coimmunoprecipitation assays in infected 293T cells and results showed that endogenous Hu-hnRNPM could specifically associate with viral vRNP(WSN).We further explored that Hu-hnRNPM could interact with NP in an RNA-independent manner.We mapped the domain/motif of Hu-hnRNPM that is responsible for NP association by in vitro GST pulldown assay and found the C-terminal domain(280-653)is critical for mediating Hu-hnRNPM-NP associations.We also examined the role of hnRNPM on vRNP nuclear-cytoplasm transport by Immunofluorescence and RNA-FISH analyses.Results showed that Hu-hnRNPM knockdown could lead to the nuclear retention of vRNP.We exclude the possibility that Hu-hnRNPM might participate in splicing of the M or NS segment.Further experiment is required to examine whether Hu-hnRNPM-NP interaction may facilitate the formation of vRNP nuclear export complex.In general,our results show that species-specific difference in hnRNPMs affects its ability to facilitate influenza A virus replication.We showed that Hu-hnRNPM could positively regulate influenza A virus RNA synthesis and is responsible for efficient viral vRNP nuclear export.These results not only provide a clearer understanding of the molecular biological mechanisms that cause influenza virus polymerase host specificity,but also indicate that Hu-hnRNPM supports the interspecies transmission from avian to human,but not from human to avian.The interaction between Hu-hnRNPM and vRNPs could be a potential target for antiviral developments.