Background::During hepatitis B virus (HBV) infection, virus-infected hepatocytes directly cross-present viral antigens and regulate T cell response within the liver microenvironment. However, little is known regarding the regulatory pathways involved in viral antigen presentation in HBV-infected hepatocytes. This study investigated the underlying mechanism of antigen assembly and the HBV antigen-presenting function of major histocompatibility complex (MHC) class I molecules using heat shock protein gp96.Methods::First, western blotting, flow cytometry, co-immunoprecipitation, GST pull-down, and confocal microscopic assays were performed to determine whether endogenous gp96 affects MHC-I levels via an antigen presentation pathway. Second, the B3Z assay and an AAV/HBV-infected hepatocyte-specific gp96-deficient mouse model were used to determine whether gp96 knockout functionally impaired peptide cross-presentation and produced a weakened antiviral cytotoxic T cell (CTL) response both in vivo and in vitro. Finally, confocal microscopic analysis and the B3Z assay were employed to show that exogenous gp96-associated peptide was present in MHC-I molecules via the endoplasmic reticulum (ER)-Golgi secretory pathway. Results::Compared with the control, gp96 knockdown significantly reduced the cell surface levels of MHC-I by approximately 75% ( P < 0.01). Endogenous gp96 interacts with MHC-I and is involved in antigen presentation. Moreover, a weakened antiviral CTL response (34% compared to control mice) has been observed in hepatocyte-specific gp96-deficient mice following HBV infection. gp96 directed exogenous antigen to the ER, and the exogenous gp96-chaperoned peptide was endosome- and proteasome-dependent but not transporter associated with antigen processing dependent. Conclusions::Cellular gp96 promotes the assembly and antigen presentation of MHC class I molecules. In addition, extracellular gp96 served as a natural adjuvant to induce a CTL response in a concerted and regulated manner within different cellular compartments. Our results elucidate the mechanism of assembly of MHC class I molecules by gp96, which may be beneficial for the design of immunotherapy and vaccines.

Hepatitis B virus;Antigen cross-presentation;Cytotoxic T-cell;gp96

Qin Lijuan;Liu Yongai;Xu Yuxiu;Li Yang;Hu Jun;Ju Ying;Zhang Yu;Wang Shuo;Li Zihai;Li Changfei;Li Xin;Meng Songdong

CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Center for Biosafety Mega-Science, Chinese Academy of Sciences (CAS), Beijing 100101, China;University of Chinese Academy of Sciences, Beijing 100190, China;Ohio State University, Ohio, USA

感染性疾病和免疫（英文）

[1]Qin Lijuan;Liu Yongai;Xu Yuxiu;Li Yang;Hu Jun;Ju Ying;Zhang Yu;Wang Shuo;Li Zihai;Li Changfei;Li Xin;Meng Songdong-.MHC Class I Assembly Function and Intracellular Transport Routes for Hepatitis B Virus Antigen Cross-presentation by Heat Shock Protein gp96)[J].感染性疾病和免疫（英文）,2022(03):183-192

Routes,B3Z,chaperoned

MHC,Class,Assembly,Function,Intracellular,Transport,Hepatitis,Virus,Antigen,Cross,presentation,by,Heat,Shock,Protein,gp96,Background,During,hepatitis,virus,HBV,infection,infected,hepatocytes,directly,cross,antigens,response,within,liver,microenvironment,However,little,known,regarding,regulatory,pathways,involved,This,study,investigated,underlying,mechanism,assembly,presenting,major,histocompatibility,complex,class,molecules,using,heat,shock,protein,Methods,First,western,blotting,flow,cytometry,immunoprecipitation,GST,pull,confocal,microscopic,assays,were,performed,determine,whether,endogenous,affects,levels,via,Second,AAV,specific,deficient,mouse,model,used,knockout,functionally,impaired,peptide,produced,weakened,antiviral,cytotoxic,CTL,both,vivo,vitro,Finally,analysis,employed,show,that,exogenous,associated,was,endoplasmic,reticulum,ER,Golgi,secretory,Results,Compared,control,knockdown,significantly,reduced,surface,approximately,Endogenous,interacts,Moreover,compared,mice,has,been,observed,following,directed,endosome,proteasome,dependent,but,transporter,processing,Conclusions,Cellular,promotes,addition,extracellular,natural,adjuvant,induce,concerted,regulated,manner,different,compartments,Our,results,elucidate,which,may,beneficial,design,immunotherapy,vaccines,Cytotoxic

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