title: Functional characterization of hepatitis B virus core promoter mutants revealed transcriptional interference among co-terminal viral mRNAs.
abstract: Hepatitis B virus (HBV) has a 3.2 kb circular DNA genome. It employs four promoters in conjunction with a single polyadenylation signal to generate 3.5, 2.4, 2.1 and 0.7 kb co-terminal RNAs. The 3.5 kb RNA is subdivided into the precore RNA for e-antigen expression and pregenomic RNA for genome replication. When introduced to a genotype A clone, several core promoter mutations markedly enhanced HBV genome replication, but suppressed e-antigen expression through up-regulation of pregenomic RNA at the expense of precore RNA. In this study, we found such mutations also diminished envelope proteins and hepatitis B surface antigen, products of the 2.1 and 2.4 kb subgenomic RNAs. Indeed, Northern blot analysis revealed overall increase in 3.5 kb RNA, but reduction in all subgenomic RNAs. To validate transcriptional interference, we subcloned 1.1×, 0.7× and 0.6× HBV genome, respectively, to a vector with or without a cytomegalovirus (CMV) promoter at the 5' end, so as to produce the pregenomic RNA, 2.4 kb RNA, and 2.1 kb RNA in large excess or not at all. Parallel transfection of the three pairs of constructs into a human hepatoma cell line confirmed the ability of pregenomic RNA to suppress all subgenomic transcripts and established the ability of the 2.4 and 2.1 kb RNAs to suppress the 0.7 kb RNA. Consistent with our findings, pregenomic RNA of the related duck HBV has been reported to interfere with transcription of the subgenomic RNAs. Transcriptional interference might explain why HBV produces so little 0.7 kb RNA and HBx protein despite a strong X promoter.
authors: Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, PR China.
authors: Chaoyang
authors: C
authors: Chen
authors: Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, PR China.
authors: Haodi
authors: H
authors: Jia
authors: Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, PR China.
authors: Fei
authors: F
authors: Zhang
authors: Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, PR China.
authors: Yanli
authors: Y
authors: Qin
authors: Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, PR China.
authors: Li
authors: L
authors: Zong
authors: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, PR China.
authors: Quan
authors: Q
authors: Yuan
authors: Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, PR China.
authors: Yongxiang
authors: Y
authors: Wang
authors: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, PR China.
authors: Ningshao
authors: N
authors: Xia
authors: The Liver Research Center, Rhode Island Hospital, Warren Alpert School of Medicine, Brown University, Providence, RI, USA.
authors: Jisu
authors: J
authors: Li
authors: Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, PR China.
authors: Yumei
authors: Y
authors: Wen
authors: The Liver Research Center, Rhode Island Hospital, Warren Alpert School of Medicine, Brown University, Providence, RI, USA.|Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, PR China.
authors: Shuping
authors: S
authors: Tong
mesh_terms: D015967:Gene Expression Regulation, Viral; D006509:Hepatitis B / Q000821:virology*; D006512:Hepatitis B Core Antigens / Q000235:genetics / Q000378:metabolism; D006514:Hepatitis B Surface Antigens / Q000235:genetics / Q000378:metabolism; D006513:Hepatitis B e Antigens / Q000235:genetics / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D006512:Hepatitis B Core Antigens; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D012333:RNA, Messenger; D012367:RNA, Viral