Scavenger Receptor A Negatively Regulates HBV Induced MDA5/RIG-Ⅰ Signaling Pathway By Inhibiting TRAF3 Ubiquitination

Hepatitis B Virus(HBV)infection is a worldwide challenge,leading to cirrhosis and hepatocellular carcinoma with its chronic infection.HBV infection is a sophisticated progress owing to various factors,such as genotypes,replication levels,and mutation sites of the virus,or the gender and age of the infected people.The immune response against HBV plays a critical role in this process,as depressed response typically leading to the chronicity of the virus.But the detailed mechanism remained unknown.Therefore,exploring the occurrence and development of the HBV infection,and how the host immune system fights against it would provide the precondition for efficient treatment and intervening strategies of hepatitis B.Scavenger receptor A(SRA,also called CD204)is one of the most important pattern recognition receptors(PRRs)in innate immune system,expressing mainly on the surface of macrophages in different tissues,especially on macrophages in lamina propria mucosae of gastrointestinal tract,kupffer cells in liver and alveolar macrophages in lung.In our previous studies,we found that SRA could also present in hepatocytes and even plays a critical role in maintaining the microenvironmental homeostasis in liver.Besides,SRA is abundantly expressed in hepatic tissue and serum of patients with hepatitis B;soluble SRA could weaken CD8+ T cell response against HBV.In spite of increasing researches suggesting that SRA plays a role in immunoregulation,the studies focusing on SRA modulating immune response against viruses are rare,and there has no report about that against HBV infection.HBV DNA and RNA can be recognize by many kinds of DNA and RNA sensors in cells during its replication,the signal then is conducted by the downstream chains consist of a net of molecules,leading to the production of type I and I interferon(IFN).In this study,we focused on main sensors of viral RNA retinoic acid-inducible gene-I(RIG-I)and melanoma differentiation associated gene-5(MDA5).TNF receptor-associated factor 3(TRAF3)is a key adaptor protein in signaling pathways of innate immunity fighting against viruses.Both RIG-I and MDA5 in cells can recruit signal molecules IPS-1,TRAF3 and TBK1 after recognizing ssRNA and dsRNA of invading virus,prompting the expression of type I IFN coordinately.In this study,we built the HBV-infected model by infecting C57BL/6 mice with pHBV1.3 plasmids through high-pressure intravenous injection.Compared with wild type(WT)mice,SRA-deficient(SRA-/-)mice had a enhanced clearing ability of HBV antigen HBsAg,HBeAg and HBcAg,and increased production of IFN-a and TNF-a,but the replication of HBV was depressed.Simulating the infection of HBV,we chose the cell line Hep G2.2.15 or infected Hep 2 with pHBV1.3 plasmids using PEI in vitro.The immune response in cells with SRA expression knocked down was enhanced compared with that in cells with normal SRA expression.The results were consistent with those in vivo.These results suggesting that SRA might negatively regulate the innate immunoresponse against HBV.In order to explore the molecular mechanism,we determined the regulation of SRA to MDA5/RIG-I pathway.The results showed that SRA could inhibit the phosphorylation and nuclear translocation of IRF3 and IRF7.Furthermore,we found that SRA could interact with TARF3 directly,and inhibited its K63-linked ubiquitination,thus restrained the anti-viral signal from conduction,leading to the attenuate phosphorylation and nuclear transfer and decreased expression of genes related to innate immunity.As SRA has no deubiquitinating activity,we explored several deubiquitinating enzymes and found that OTUB1 can be recruited by SRA and interact with each other,thus resulted in the inhibition of K63-linked ubiquitination of TRAF3.Taken together,SRA can interact with TRAF3 directly,and inhibit its K63-linked ubiquitination by recruiting OTUB1,leading to repression of phosphorylation and nuclear transfer of IRF3 and IRF7,which supress the expression of genes related to innate immunoresponse induced by HBV.Therefore,SRA may be taken as a new target of immnotherapeutic strategy for Hepatic B.