Doubly Spliced RNA of Hepatitis B Virus Suppresses Viral Transcription via TATA-Binding Protein and Induces Stress Granule Assembly.
The risk of liver cancer in patients infected with the hepatitis B virus (HBV) and their clinical response to interferon alpha therapy vary based on the HBV genotype. The mechanisms underlying these differences in HBV pathogenesis remain unclear. In HepG2 cells transfected with a mutant HBV(G2335A) expression plasmid that does not transcribe the 2.2-kb doubly spliced RNA (2.2DS-RNA) expressed by wild-type HBV genotype A, the level of HBV pregenomic RNA (pgRNA) was higher than that in cells transfected with an HBV genotype A expression plasmid. By using cotransfection with HBV genotype D and 2.2DS-RNA expression plasmids, we found that a reduction of pgRNA was observed in the cells even in the presence of small amounts of the 2.2DS-RNA plasmid. Moreover, ectopic expression of 2.2DS-RNA in the HBV-producing cell line 1.3ES2 reduced the expression of pgRNA. Further analysis showed that exogenously transcribed 2.2DS-RNA inhibited a reconstituted transcription in vitro. In Huh7 cells ectopically expressing 2.2DS-RNA, RNA immunoprecipitation revealed that 2.2DS-RNA interacted with the TATA-binding protein (TBP) and that nucleotides 432 to 832 of 2.2DS-RNA were required for efficient TBP binding. Immunofluorescence experiments showed that 2.2DS-RNA colocalized with cytoplasmic TBP and the stress granule components, G3BP and poly(A)-binding protein 1 (PABP1), in Huh7 cells. In conclusion, our study reveals that 2.2DS-RNA acts as a repressor of HBV transcription through an interaction with TBP that induces stress granule formation. The expression of 2.2DS-RNA may be one of the viral factors involved in viral replication, which may underlie differences in clinical outcomes of liver disease and responses to interferon alpha therapy between patients infected with different HBV genotypes.


OBJECTIVE
Patients infected with certain genotypes of HBV have a lower risk of hepatocellular carcinoma and exhibit a more favorable response to antiviral therapy than patients infected with other HBV genotypes. Using cultured human hepatoma cells as a model of HBV infection, we found that the expression of 2.2DS-RNA caused a decrease in HBV replication. In cultured cells, the ectopic expression of 2.2DS-RNA obviously reduced the intracellular levels of HBV mRNAs. Our analysis of the 2.2DS-RNA-mediated suppression of viral RNA expression showed that 2.2DS-RNA inhibited transcription via binding to the TATA-binding protein and stress granule proteins. Our findings suggest that the 2.2DS-RNA acts as a suppressive noncoding RNA that modulates HBV replication, which may in turn influence the development of chronic hepatitis B.
Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan.
Kuen-Nan
KN
Tsai
Faculty of Traditional Chinese Medicine, Southern University College, Skudai, Johor, Malaysia.
Chin-Liew
CL
Chong
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.
Yu-Chi
YC
Chou
Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan.
Chien-Chiao
CC
Huang
Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan Institute of Microbiology and Immunology, National Yang Ming University, Taipei, Taiwan.
Yi-Ling
YL
Wang
Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan.
Shao-Win
SW
Wang
Center for Molecular Medicine, China Medical University and Hospital, Taichung, Taiwan.
Mong-Liang
ML
Chen
Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan chunhong@nhri.org.tw tonychang@nhri.org.tw.
Chun-Hong
CH
Chen
Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan Institute of Microbiology and Immunology, National Yang Ming University, Taipei, Taiwan chunhong@nhri.org.tw tonychang@nhri.org.tw.
Chungming
C
Chang
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000453:epidemiology / Q000821:virology; D002352:Carrier Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D004265:DNA Helicases; D015967:Gene Expression Regulation, Viral; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006514:Hepatitis B Surface Antigens / Q000378:metabolism; D006513:Hepatitis B e Antigens / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000821:virology; D006801:Humans; D008113:Liver Neoplasms / Q000453:epidemiology / Q000821:virology; D051379:Mice; D010957:Plasmids / Q000235:genetics; D039122:Poly(A)-Binding Protein I / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D020365:RNA Helicases; D012322:RNA Precursors / Q000096:biosynthesis; D000067776:RNA Recognition Motif Proteins; D012326:RNA Splicing / Q000235:genetics; D012330:RNA, Double-Stranded / Q000096:biosynthesis / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics; D016601:RNA-Binding Proteins / Q000378:metabolism*; D035181:TATA-Box Binding Protein / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D014779:Virus Replication / Q000235:genetics*
D002352:Carrier Proteins; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D039122:Poly(A)-Binding Protein I; D000075223:Poly-ADP-Ribose Binding Proteins; D012322:RNA Precursors; D000067776:RNA Recognition Motif Proteins; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D035181:TATA-Box Binding Protein; D004265:DNA Helicases; C116070:G3BP1 protein, human; D020365:RNA Helicases