Doubly Spliced RNA of Hepatitis B Virus Suppresses Viral Transcription via TATA-Binding Protein and Induces Stress Granule Assembly

J Virol. 2015 Nov 15;89(22):11406-19. doi: 10.1128/JVI.00949-15. Epub 2015 Sep 2.

Doubly Spliced RNA of Hepatitis B Virus Suppresses Viral

Transcription via TATA-Binding Protein and Induces Stress

Granule Assembly.

Tsai KN1_{, Chong CL}2_{, Chou YC}3_{, Huang CC}4_{, Wang YL}5_{, Wang SW}6_{, Chen ML}7_{, Chen CH}8_,

Chang C9_.

Author information

 1_{Institute of Molecular and Genomic Medicine, National Health Research Institutes,}

Miaoli, Taiwan Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan.

 2_{Faculty of Traditional Chinese Medicine, Southern University College, Skudai, Johor,}

Malaysia.

 3_{Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.}

 4_{Department of Biomedical Science and Environmental Biology, Kaohsiung Medical}

University, Kaohsiung, Taiwan.

 5_{Institute of Molecular and Genomic Medicine, National Health Research Institutes,}

Miaoli, Taiwan Institute of Microbiology and Immunology, National Yang Ming University, Taipei, Taiwan.

 6_{Institute of Molecular and Genomic Medicine, National Health Research Institutes,}

Miaoli, Taiwan.

 7_{Center for Molecular Medicine, China Medical University and Hospital, Taichung,}

Taiwan.

 8_{Institute of Molecular and Genomic Medicine, National Health Research Institutes,}

Miaoli, Taiwan chunhong@nhri.org.tw tonychang@nhri.org.tw.

 9_{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.

Abstract

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.

IMPORTANCE:

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.