Lijuan Qin, Yongai Liu, Yuxiu Xu, Yang Li, Jun Hu, Y. Ju, Yu Zhang, Shuo Wang, Zihai Li, Changfei Li, Xin Li, S. Meng
{"title":"热休克蛋白gp96对乙型肝炎病毒抗原交叉呈递的MHC I类组装功能和细胞内转运途径","authors":"Lijuan Qin, Yongai Liu, Yuxiu Xu, Yang Li, Jun Hu, Y. Ju, Yu Zhang, Shuo Wang, Zihai Li, Changfei Li, Xin Li, S. Meng","doi":"10.1097/ID9.0000000000000058","DOIUrl":null,"url":null,"abstract":"Abstract 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.","PeriodicalId":73371,"journal":{"name":"Infectious diseases & immunity","volume":"2 1","pages":"183 - 192"},"PeriodicalIF":0.0000,"publicationDate":"2022-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"2","resultStr":"{\"title\":\"MHC Class I Assembly Function and Intracellular Transport Routes for Hepatitis B Virus Antigen Cross-presentation by Heat Shock Protein gp96\",\"authors\":\"Lijuan Qin, Yongai Liu, Yuxiu Xu, Yang Li, Jun Hu, Y. Ju, Yu Zhang, Shuo Wang, Zihai Li, Changfei Li, Xin Li, S. Meng\",\"doi\":\"10.1097/ID9.0000000000000058\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Abstract 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.\",\"PeriodicalId\":73371,\"journal\":{\"name\":\"Infectious diseases & immunity\",\"volume\":\"2 1\",\"pages\":\"183 - 192\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2022-07-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"2\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Infectious diseases & immunity\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1097/ID9.0000000000000058\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Infectious diseases & immunity","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1097/ID9.0000000000000058","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
MHC Class I Assembly Function and Intracellular Transport Routes for Hepatitis B Virus Antigen Cross-presentation by Heat Shock Protein gp96
Abstract 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.