BAG6 Negatively Regulates RNA Virus-mediated Innate Immune Signaling Pathway By Targeting VISA

The innate immune system is the first line of defense against the invasion of exogenous microorganisms.After pathogenic microorganisms infect host,the host cells recognize the conserved component pathogen-associated molecular patterns(PAMPs)released into the cell by the pathogenic microorganism through its pattern recognition receptors(PRRs),which then initiate a signaling cascade to induce the organism to produce Type I interferon and proinflammatory cytokine and promote the expression of antiviral genes to clear the invading pathogen to maintain homeostasis.After RNA viruses invade the host cells,intracellular RIG-Ⅰ-like receptors(RLRs)recognize viral ds RNA.RLRs mainly include RIG-Ⅰ and MDA5,with RIG-Ⅰ tending to recognize 5’-ppp ds RNA and short double-stranded RNA(short ds RNA)and MDA5 tending to recognize long double-stranded RNA(long ds RNA),both of which have VISA(also known as MAVS,ISP-1,and Cardif)as a downstream adaptor protein.As an antiviral signaling protein localized on mitochondria,VISA has an irreplaceable role in innate immune signaling.Although a lot of studies on the regulatory mechanisms of VISA-mediated signaling have been published in recent years,the signaling regulatory network of VISA is still not well established.Our laboratory,as one of the discoverers of the VISA protein,further obtained a series of candidate proteins including BAG6 by large-scale yeast two-hybrid screening using VISA as a bait protein.Subsequent studies revealed that BAG6 suppressed RNA virus-induced activation of IFN-β promoter,ISRE,and NF-κB reporter genes.qPCR experiments showed that BAG6 was able to suppress Sendai virus(SeV)-induced transcript levels of downstream antiviral genes.Native PAGE and immunoblotting experiments showed that BAG6 suppressed SeV-induced IRF3 dimerization and phosphorylation of TBK1,IRF3,and P65.Subsequently,we generated BAG6-deficient cell lines in HEK 293,HeLa,NIH3T3,and mouse primary bone marrow-derived macrophages(BMDMs)using gene editing technology(CRISPR-Cas9)and subsequently performed all the above experiments with corresponding results,and plaque assays showed that knockout of BAG6 significantly promoted the cells’ antiviral activity.We further explored the regulatory mechanism of BAG6 in RIG-Ⅰ/VISA-mediated signal transduction.Immunoprecipitation experiments showed that BAG6 specifically interacted with VISA and that the interaction was regulated by the virus.In subsequent experiments,we found that BAG6 promoted K48-polyubiquitination of VISA and reduced VISA’s oligomerization to suppress the signaling.In addition,BAG6 attenuated the recruitment of VISA to the downstream molecule TRAF2 and promoted K48-polyubiquitination of TRAF2 to inhibit RNA virus-induced innate immune signaling,and these experimental results were validated in multiple BAG6-deficient cell lines.In this study,we confirmed that BAG6 negatively regulates antiviral innate immune signaling by targeting the mitochondrial antiviral signaling protein VISA,both at the cellular level and mouse primary immune cell level,which further completes the mechanism of VISA-mediated antiviral signaling pathway and provides a new theoretical basis and molecular target for future biomedical research and disease prevention.