| H9N2 is one of the major subtypes influenza virus circulating in poultry in China,which has a wide infection host range from bird to mammals.Two H9N2 viruses were isolated from one mink farm in 2014,named A/mink/China/01/2014 and A/mink/China/02/2014.Phylogenetic analysis showed that internal genes of the two H9N2 viruses have close relationship with that of H7N9 viruses.Interestingly,two H9N2 separated in phylogenetic trees,indicating that they are introduced to this mink farms in two independent events.And further mice studies showed that A/mink/China/01/2014 caused obvious weight loss and 20%mortality in infected mice,while another virus did not cause any disease in mice infected at a same dose.Genetic analysis indicated that the virulent H9N2 contain a naturel mutation at701 N in PB2 protein,which was reported to contribute to mammalian adaptation.Circulation of H9N2 in mink may drive the virus to adapt mammals and continual surveillance of influenza virus in mink was warranted.To further study the interaction between A/mink/China/01/2014 of H9N2,H1N1 and the infected cells,the RNA-Seq technology was used to analyze the infected cells’ mRNA transcriptome.Finally,compared to the uninfected group,we got 371 differentially expressed genes in H9N2 group,with 141 up-regulated and 230 down-regulated genes;and in H1N1 group,1116 differentially expressed genes were found including 360 up-regulated and 756down-regulated genes.According to KEGG annotation,the differentially expressed genes were significantly enriched in the signal transduction,followed by endocrine system and immune system.Influenza virus replication took place within the nucleus,and the stead system will be broken when a large number of viral nucleic acids were synthesized in the nucleus that will cause the levels of some proteins increase or decline.After infected by H9N2 or H1N1 influenza virus,there were two increasing proteins and seven reducing protein in hnRNP polycomb group proteins,which included hnRNP H1 protein.HnRNP H1 protein,one of the abundant proteins in eukaryotic cells,plays an important role in pre-mRNA cutting,processing and transportation of the host cells.After mRNA transcription analysis,the level of hnRNP H1 protein mRNA was decline after the cells infected with influenza virus,and the result of Real-Time PCR was the same as the result ofm RNA transcription analysis after the cells infected with WSN virus.In order to study whether hnRNP H1 protein influences influenza virus replication or not,the study overexpressed hnRNP H1 protein in cells and found that the replication of influenza virus was inhibited,similarly,the si-hnRNP H1 protein cells could promote the influenza virus replication.To further explore the mechanism of the inhibition effect of hnRNP H1 protein on influenza virus replication,the luciferase assay was designed and the result demonstrated that hnRNP H1 protein had inhibition effect on polymerase activity of influenza virus.HnRNP H1 protein is an RNA binding protein that may bind influenza virus RNA to inhibit viral replication.To verify the guess,hnRNP H1 protein was induced expression and purified and then the electrophoretic mobility shift assay was designed and the result found that hnRNP H1 protein could bind polymerase proteins vRNA of influenza virus.In order to explore the functional region of hnRNP H1 protein,the protein was truncated and mutant to test their influences on polymerase activity of influenza virus,both with the co-immunoprecipitation assay results denoted that the functional region of hnRNP H1 protein was in the N-terminal and the main amino acid sites are tryptophan,tyrosine in RRM1 region and the phenylalanine,tyrosine in RRM2 region.Above all,the main four amino acid sites of hnRNP H1 protein could bind with the-GGGG-nucleic acid regions of influenza virus to inhibit the replication of influenza virus for combatting influenza virus infection.What’s more,hnRNPH family proteins have the function of inhibiting influenza virus replicaion. |
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