Hepatitis B virus nuclear export elements: RNA stem-loop α and β, key parts of the HBV post-transcriptional regulatory element

ABSTRACT Hepatitis B virus (HBV) RNA is downregulated by inflammatory cytokines induced in the liver by adoptively transferred HBV-specific cytotoxic T lymphocytes (CTLs) and during murine cytomegalovirus (MCMV) infections of the livers of HBV transgenic mice. The disappearance of HBV RNA is tightly associated with the cytokine-induced proteolytic cleavage of a previously defined HBV RNA-binding protein known as La autoantigen. La binds to a predicted stem-loop structure at the 5′ end of the posttranscriptional regulatory element of HBV RNA between nucleotides 1243 and 1333. In the present study, we searched for nuclear RNase activities that might be involved in HBV RNA decay. Nuclear extracts derived from control livers and CTL-injected and MCMV-infected livers were analyzed for the ability to cleave HBV RNA. Endonucleolytic activity that cleaved HBV RNA at positions 1269 to 1270 and 1271 to 1272, immediately 5′ of the stem-loop bound by the La protein (positions 1272 to 1293), was detected. Furthermore, we provide evidence that the cytokine-dependent downregulation of HBV RNA following MCMV infection is temporally associated with the upregulation of the endonucleolytic activity herein described. Collectively, these results suggest a model in which the steady-state HBV RNA content is controlled by the stabilizing influence of La and the destabilizing influence of nuclear RNase activities.

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Inhibition of Hepatitis B Virus Replication by MyD88 Involves Accelerated Degradation of Pregenomic RNA and Nuclear Retention of Pre-S/S RNAs

Journal of Virology ◽

10.1128/jvi.00236-10 ◽

2010 ◽

Vol 84 (13) ◽

pp. 6387-6399 ◽

Cited By ~ 49

Author(s):

Jianhua Li ◽

Shanshan Lin ◽

Qiying Chen ◽

Lu Peng ◽

Jianwei Zhai ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Antiviral Activity ◽

Nuclear Export ◽

Regulatory Element ◽

Primary Response ◽

Proximal Region ◽

Hbv Replication ◽

Polypyrimidine Tract Binding Protein ◽

B Virus

ABSTRACT Myeloid differentiation primary response protein 88 (MyD88), which can be induced by alpha interferon (IFN-α), has an antiviral activity against the hepatitis B virus (HBV). The mechanism of this antiviral activity remains poorly understood. Here, we report that MyD88 inhibited HBV replication in HepG2.2.15 cells and in a mouse model. The knockdown of MyD88 expression weakened the IFN-α-induced inhibition of HBV replication. Furthermore, MyD88 posttranscriptionally reduced the levels of viral RNA. Remarkably, MyD88 accelerated the decay of viral pregenomic RNA in the cytoplasm. Mapping analysis showed that the RNA sequence located in the 5′-proximal region of the pregenomic RNA was critical for the decay. In addition, MyD88 inhibited the nuclear export of pre-S/S RNAs via the posttranscriptional regulatory element (PRE). The retained pre-S/S RNAs were shown to degrade in the nucleus. Finally, we found that MyD88 inhibited the expression of polypyrimidine tract-binding protein (PTB), a key nuclear export factor for PRE-containing RNA. Taken together, our results define a novel antiviral mechanism against HBV mediated by MyD88.

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Woodchuck Hepatitis Virus Contains a Tripartite Posttranscriptional Regulatory Element

Journal of Virology ◽

10.1128/jvi.72.6.5085-5092.1998 ◽

1998 ◽

Vol 72 (6) ◽

pp. 5085-5092 ◽

Cited By ~ 222

Author(s):

John E. Donello ◽

Jonathan E. Loeb ◽

Thomas J. Hope

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Hepatitis Virus ◽

Regulatory Element ◽

Deletion Analysis ◽

Woodchuck Hepatitis Virus ◽

B Virus ◽

Cytoplasmic Accumulation ◽

Posttranscriptional Regulatory Element

ABSTRACT The hepatitis B virus posttranscriptional regulatory element (HBVPRE) is a cis-acting RNA element that partially overlaps with enhancer I and is required for the cytoplasmic accumulation of HBV surface RNAs. We find that the closely related woodchuck hepatitis virus (WHV), which has been shown to lack a functional enhancer I, also contains a posttranscriptional regulatory element (WPRE). Deletion analysis suggests that the WPRE consists of three independent subelements. Comparison of the bipartite HBVPRE and tripartite WPRE activities reveals that the tripartite WPRE is two to three times more active than the bipartite HBVPRE. Mutation of a single WPRE subelement decreases WPRE activity to the level of the HBVPRE. Bipartite and tripartite chimeras of the WPRE and HBVPRE possess activities which suggest that elements containing three subelements are posttranscriptionally stronger than those containing two. These data demonstrate that the posttranscriptional regulatory element is conserved within the mammalian hepadnaviruses and that its strength is determined by the number of subelements within the RNA.

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N6-methyladenosine modification of hepatitis B virus RNA differentially regulates the viral life cycle

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1808319115 ◽

2018 ◽

Vol 115 (35) ◽

pp. 8829-8834 ◽

Cited By ~ 44

Author(s):

Hasan Imam ◽

Mohsin Khan ◽

Nandan S. Gokhale ◽

Alexa B. R. McIntyre ◽

Geon-Woo Kim ◽

...

Keyword(s):

Life Cycle ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Reverse Transcription ◽

Mutational Analysis ◽

Regulatory Function ◽

Messenger Rnas ◽

Stem Loop ◽

B Virus ◽

A Site

N6-methyladenosine (m6A) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found m6A on both cellular and viral transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that m6A and its associated machinery regulate the life cycle of hepatitis B virus (HBV). HBV is a DNA virus that completes its life cycle via an RNA intermediate, termed pregenomic RNA (pgRNA). Silencing of enzymes that catalyze the addition of m6A to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the m6A site in the HBV RNA and found that a conserved m6A consensus motif situated within the epsilon stem loop structure, is the site for m6A modification. The epsilon stem loop is located in the 3′ terminus of all HBV mRNAs and at both the 5′ and 3′ termini of the pgRNA. Mutational analysis of the identified m6A site in the 5′ epsilon stem loop of pgRNA revealed that m6A at this site is required for efficient reverse transcription of pgRNA, while m6A methylation of the 3′ epsilon stem loop results in destabilization of all HBV transcripts, suggesting that m6A has dual regulatory function for HBV RNA. Overall, this study reveals molecular insights into how m6A regulates HBV gene expression and reverse transcription, leading to an increased level of understanding of the HBV life cycle.

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La Autoantigen Specifically Recognizes a Predicted Stem-Loop in Hepatitis B Virus RNA

Journal of Virology ◽

10.1128/jvi.73.7.5767-5776.1999 ◽

1999 ◽

Vol 73 (7) ◽

pp. 5767-5776 ◽

Cited By ~ 63

Author(s):

Tilman Heise ◽

Luca G. Guidotti ◽

Francis V. Chisari

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Rna Stability ◽

Dependent Manner ◽

Necrosis Factor Alpha ◽

Stem Loop ◽

Stem Loop Structure ◽

La Protein ◽

Virus Rna ◽

B Virus

ABSTRACT We recently identified three nuclear proteins (p45, p39, and p26) that bind to a 91-nucleotide (nt) RNA element between nt 1243 and 1333 in hepatitis B virus (HBV) RNA, and we showed that these proteins and HBV RNA are regulated coordinately by gamma interferon and tumor necrosis factor alpha. Purification and sequence analysis of tryptic peptides obtained from p39 revealed sequence homology to the mouse La protein. Immunoprecipitation experiments showed that p45, p39, and p26 were recognized by anti-La-specific antiserum, indicating that p45 is the full-length La protein and that p39 and p26 are likely to be proteolytic La cleavage products. Furthermore, in competition experiments we found that all three La proteins bind, in a phosphorylation-dependent manner, to the same predicted stem-loop structure located between nt 1275 and 1291 of HBV, withKd s of approximately 1.0 nM. Collectively, these results support the notion that the La protein may contribute to HBV RNA stability, constitutively and in response to inflammatory cytokines.

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