Molecular Mechanism Of Host Factor ZFAT Silencing Inhibited Replication Of H5N1 Highly-pathogenic Avian Influenza Virus

H5N1 highly pathogenic avian influenza(HPAIV)has been a long-term threat to animal and human health,seasonal outbreak was often triggered by cross-species spreading.H5N1 highly pathogenic avian influenza virus(HPAIV)replicated in mammals relying on the sophisticated biological processes involving viral nucleoprotein complex(vRNP),meanwhile,viral non-structural protein 1(NS1)can promote replication of influenza virus through innate immune antagonism and viral polymerase enhancement performed by interactions with various host factors.Zincfinger and AT hook domain containing protein(ZFAT)is a member of zinc-finger protein family,regulating the hematopoietic differentiation and maintaining peripheral leukocytes homeostasis,ZFAT is associated with transcriptional regulation of immune genes,while no reports about anti-virus immune involving ZFAT have been published.Clustered regularly interspaced short palindromic repeats(CRISPR)and CRISPR associated protein 9(Cas9)gene-editing system was applied to construct ZFAT knock-out cell line,which is A549-CRISPR-Cas9-ZFAT-KO.By utilizing replicating titration assay,viral replication of H5N1 HPAIV was tested on A549-CRISPR-Cas9-ZFAT-KO cell line,leaser confocal microscopy was applied to exhibit space distribution of ZFAT during viral infection,revealed the effect to infection processes induced by ZFAT silencing,and the efficiency of nuclear-importing and assembling of vRNP was performed by nuclear extraction assay.Overexpression vectors for ZFAT and viral proteins were constructed for pathogen-host interaction,CoImmunoprecipitation(Co-IP)assay was applied for testing interactions between cotransfected ZFAT and other viral proteins.Cellular sites of interaction were performed by confocal microscopy for co-transfected cell,providing evidences for further discussion of functions and molecular mechanism induced by ZFAT silencing during viral infection.Those experimental results demonstrated that ZFAT silencing can inhibit viral replication and delay vRNP nuclear importing,decreasing the efficiency of vRNP assembling in nuclear,moreover,ZFAT overlapped with vRNP in space distribution during nuclear importing,Co-IP and following confocal microscopy for cotransfected cell indicated that ZFAT interacted with viral NS1 protein in nuclear,mutation of K41 amino acid site in NS1 protein eliminated the interaction with ZFAT in nuclear.ZFAT may play a role in NS1 relevant functions during H5N1 HPAIV infection,silencing of ZFAT expression may enhance host resistance to viral infection.