Mechanism Of GATA3 Anti-influenza A Virus Replication And Its Pan-cancer Bioinformatics Analysis

Influenza pandemics and seasonal influenza,which occur annually worldwide,place a significant medical and economic burden on global health systems.Influenza A virus(IAV)is currently the main virus responsible for influenza pandemics worldwide.The highly variable nature of influenza viruses,their many subtypes and the wide range of hosts make their prevention a challenge to date.In order to better prevent and treat influenza-related illnesses,we need to better understand the transcription,replication and pathogenesis of IAV.Influenza A virus infection in humans results in the production of interferons(IFNs)by host cells in the body.Interferon signal induces the expression of hundreds of interferon stimulated genes(ISGs)through the JAK-STAT pathway,which in turn acts as an antiviral agent.Interferon resistance to influenza viruses is part of the body' s natural immunity,the innate immune system.Most of the mechanisms of ISGs against influenza viruses are still unknown.In this paper,we selected 22 ISGs of interest and constructed a knockout cell library of human lung cancer epithelial cell line A549 by CRISPR/Cas9 knockout technology based on literature research and summary of previous laboratory findings.Firstly,we used phagocytic spot assay to detect the inhibitory effect of the selected ISGs on influenza virus.22 ISGs knockout cell lines were infected with influenza virus and the supernatants were collected after 24 and 48 hours and the virus titers were measured by phagocytosis assay for statistical purposes.The statistical results revealed a significant promotion of influenza virus production by the GATA3 knockout cells.To further determine the anti-influenza function of GATA3,we confirmed the successful knockout of GATA3 using Western Blot,PCR and gene sequencing,and verified that knockout of GATA3 had no effect on cellular activity using cellular activity assays;we stimulated wild-type and GATA3 knockout cells with interferon and poly(I:C)and confirmed that GATA3 belongs to the interferon-stimulated gene.GATA3 knockout and over-expression cell lines were infected with influenza viruses,and we confirmed that knockout of GATA3 promoted influenza virus production and over-expression of GATA3 suppressed influenza virus production using flow assays,phage spot assays,RT-PCR and Western Blot.We also performed immunofluorescence confocal assays and immunofluorescence flow assays to qualitatively and quantitatively observe a significant increase in influenza virus production after GATA3 knockont.In terms of anti-influenza virus mechanism,we first performed viral Binding assays,Entry assays and Replicon assays,which showed that GATA3 exerts antiviral effects mainly by inhibiting the replication of influenza virus.We then investigated the interaction between GATA3 and influenza virus proteins through immunoprecipitation assays.The results showed that GATA3 interacted with PB2 to inhibit the binding of PB2 to PB1 and hinder the assembly of RdRp,thus exerting anti-influenza viral effects.Our truncation experiments also revealed that GA,the smallest truncator of GATA3 so far validated,exerts a stronger effect on influenza virus replication.Studies on the association between GATA3 and cancer have been reported,but a pan-cancer analysis of GATA3 has not yet been reported.Therefore,we have analyzed the clinical relevance of GATA3 to different cancers and its possible mechanisms of action in terms of differential gene expression,clinical staging of tumour patients,survival prognosis,gene mutation and tumourigenesis,immune cell infiltration relevance and gene-related cellular pathways.In summary,our viral studies identified GATA3 as a new antiviral ISG and enhanced the body' s antiviral immune response by interfering with the viral replication process,providing new clues to the direction of the antiviral mechanism of ISGs,enriching the understanding of the natural immune antiviral mechanism of action,and providing new ideas for the development of anti-influenza viral drugs with the new findings from truncated in vivo experimental studies.Our pan-cancer analysis provides a relatively comprehensive report for the mechanism of action of GATA3 in different tumors,and provides support for GATA3 to become a clinical molecular indicator in breast cancer.