Hepatitis B Virus Posttranscriptional Regulatory Element Research Articles

Viral hijacking of the TENT4-ZCCHC14 complex protects viral RNAs via mixed tailing.

TENT4 enzymes generate 'mixed tails' of diverse nucleotides at 3' ends of RNAs via nontemplated nucleotide addition to protect messenger RNAs from deadenylation. Here we discover extensive mixed tailing in transcripts of hepatitis B virus (HBV) and human cytomegalovirus (HCMV), generated via a similar mechanism exploiting the TENT4-ZCCHC14 complex. TAIL-seq on HBV and HCMV RNAs revealed that TENT4A and TENT4B are responsible for mixed tailing and protection of viral poly(A) tails. We find that the HBV post-transcriptional regulatory element (PRE), specifically the CNGGN-type pentaloop, is critical for TENT4-dependent regulation. HCMV uses a similar pentaloop, an interesting example of convergent evolution. This pentaloop is recognized by the sterile alpha motif domain-containing ZCCHC14 protein, which in turn recruits TENT4. Overall, our study reveals the mechanism of action of PRE, which has been widely used to enhance gene expression, and identifies the TENT4-ZCCHC14 complex as a potential target for antiviral therapeutics.

HBsAg mRNA degradation induced by a dihydroquinolizinone compound depends on the HBV posttranscriptional regulatory element

In pursuit of novel therapeutics targeting the hepatitis B virus (HBV) infection, we evaluated a dihydroquinolizinone compound (DHQ-1) that in the nanomolar range reduced the production of virion and surface protein (HBsAg) in tissue culture. This compound also showed broad HBV genotype coverage, but was inactive against a panel of DNA and RNA viruses of other species. Oral administration of DHQ-1 in the AAV-HBV mouse model resulted in a significant reduction of serum HBsAg as soon as 4 days following the commencement of treatment. Reduction of HBV markers in both in vitro and in vivo experiments was related to the reduced amount of viral RNA including pre-genomic RNA (pgRNA) and 2.4/2.1 kb HBsAg mRNA. Nuclear run-on and subcellular fractionation experiments indicated that DHQ-1 mediated HBV RNA reduction was the result of accelerated viral RNA degradation in the nucleus, rather than the consequence of inhibition of transcription initiation. Through mutagenesis of HBsAg gene sequences, we found induction of HBsAg mRNA decay by DHQ-1 required the presence of the HBV posttranscriptional regulatory element (HPRE), with a 109 nucleotides sequence within the central region of the HPRE alpha sub-element being the most critical. Taken together, the current study shows that a small molecule can reduce the overall levels of HBV RNA, especially the HBsAg mRNA, and viral surface proteins. This may shed light on the development of a new class of HBV therapeutics.

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

ABSTRACTMany viruses contain RNA elements that modulate splicing and/or promote nuclear export of their RNAs. The RNAs of the major human pathogen, hepatitis B virus (HBV) contain a large (~600 bases) composite cis-acting 'post-transcriptional regulatory element' (PRE). This element promotes expression from these naturally intronless transcripts. Indeed, the related woodchuck hepadnavirus PRE (WPRE) is used to enhance expression in gene therapy and other expression vectors. These PRE are likely to act through a combination of mechanisms, including promotion of RNA nuclear export. Functional components of both the HBV PRE and WPRE are 2 conserved RNA cis-acting stem-loop (SL) structures, SLα and SLβ. They are within the coding regions of polymerase (P) gene, and both P and X genes, respectively. Based on previous studies using mutagenesis and/or nuclear magnetic resonance (NMR), here we propose 2 covariance models for SLα and SLβ. The model for the 30-nucleotide SLα contains a G-bulge and a CNGG(U) apical loop of which the first and the fourth loop residues form a CG pair and the fifth loop residue is bulged out, as observed in the NMR structure. The model for the 23-nucleotide SLβ contains a 7-base-pair stem and a 9-nucleotide loop. Comparison of the models with other RNA structural elements, as well as similarity searches of human transcriptome and viral genomes demonstrate that SLα and SLβ are specific to HBV transcripts. However, they are well conserved among the hepadnaviruses of non-human primates, the woodchuck and ground squirrel.

A conserved RNA structural element within the hepatitis B virus post-transcriptional regulatory element enhance nuclear export of intronless transcripts and repress the splicing mechanism.

Hepatitis B virus (HBV) infection is a primary cause of hepatocellular carcinoma and liver cirrhosis worldwide. To develop novel antiviral drugs, a better understanding of HBV gene expression regulation is vital. One important aspect is to understand how HBV hijacks the cellular machinery to export unspliced RNA from the nucleus. The HBV post-transcriptional regulatory element (HBV PRE) has been proposed to be the HBV RNA nuclear export element. However, the function remains controversial, and the core element is unclear. This study, therefore, aimed to identify functional regulatory elements within the HBV PRE and investigate their functions. Using bioinformatics programs based on sequence conservation and conserved RNA secondary structures, three regulatory elements were predicted, namely PRE 1151-1410, PRE 1520-1620 and PRE 1650-1684. PRE 1151-1410 significantly increased intronless and unspliced luciferase activity in both HepG2 and COS-7 cells. Likewise, PRE 1151-1410 significantly elevated intronless and unspliced HBV surface transcripts in liver cancer cells. Moreover, motif analysis predicted that PRE 1151-1410 contains several regulatory motifs. This study reported the roles of PRE 1151-1410 in intronless transcript nuclear export and the splicing mechanism. Additionally, these results provide knowledge in the field of HBV RNA regulation. Moreover, PRE 1151-1410 may be used to enhance the expression of other mRNAs in intronless reporter plasmids.

A rational study for identification of highly effective siRNAs against hepatitis B virus

RNA interference (RNAi) is a powerful gene knockdown technique used for study gene function. It also potentially provides effective agents for inhibiting infectious and genetic diseases. Most of RNAi studies employ a single siRNA designing program and then require large-scale screening experiments to identify functional siRNAs. In this study, we demonstrate that an assembly of results generated from different siRNA designing programs could provide clusters of predicting sites that aided selection of potent siRNAs. Based on the clusters, three siRNA target sites were selected on a conserved RNA region of hepatitis B virus (HBV), known as HBV post-transcriptional regulatory element (HBV PRE) at nucleotide positions 1317–1337, 1357–1377 and 1644–1664. All three chosen siRNAs driven by H1 promoter were highly effective and could drastically decrease expression of HBV transcripts (core, surface and X) and surface protein without induction of interferon response and cell cytotoxicity in liver cancer cell line (HepG2). Based on prediction of secondary structures, the silencing effects of siRNAs were less effective against a loop sequence of the mRNA target with hairpin structure. In summary, we demonstrate an effectual approach for identification of functional siRNAs. Moreover, highly potent siRNAs identified here may serve as novel agents for development of nucleic acid-based HBV therapy.

Identification of an effective siRNA target site and functional regulatory elements, within the hepatitis B virus posttranscriptional regulatory element

BackgroundInfection with hepatitis B virus (HBV) is major public health concern. The limitations of available antiviral drugs require development of novel approaches to inhibit HBV replication. This study was conducted to identify functional elements and new siRNA target sites within the highly conserved regions of the 533 base post-transcriptional regulatory element (PRE) of HBV RNAs.ResultsComputational analysis of the PRE sequence revealed several conserved regulatory elements that are predicted to form local secondary structures some of these within known regulatory regions. A deletion analysis showed that sub-elements of the PRE have different effects on the reporter activity suggesting that the PRE contains multiple regulatory elements. Conserved siRNA targets at nucleotide position 1317-1337 and 1329-1349 were predicted. Although the siRNA at the position 1329-1349 had no effect on the expression of reporter gene, the siRNA target site at the position 1317-1337 was observed to significantly decrease expression of the reporter protein. This siRNA also specifically reduced the level of cccDNA in transiently HBV infected cells.ConclusionThe HBV PRE is likely to contain multiple regulatory elements. A conserved target within this region at 1317-1337 is an effective siRNA target.

Role of glyceraldehyde-3-phosphate dehydrogenase binding to hepatitis B virus posttranscriptional regulatory element in regulating expression of HBV surface antigen

The hepatitis B virus (HBV) posttranscriptional regulatory element (HPRE) has been demonstrated to facilitate the cytoplasmic localization of unspliced transcripts. One cellular protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is known to combine with this element. However, its function on HPRE remains unclear. Here, we show that recombinant GAPDH protein binds to HPRE RNA in vitro in streptavidin pull-down assays. Functional analysis demonstrated that GAPDH inhibited HPRE function in a pDM138-HPRE chloramphenicol acetyltransferase reporter assay system. Overexpression of GAPDH depressed the expression of HBs antigen, as detected both in cells transiently expressing HBs-HPRE and in HepG2.2.15 cells. These data indicate that GAPDH may be involved in the posttranscriptional regulation of HBV, which is critical for the life cycle of HBV.

Solution structure of stem-loop α of the hepatitis B virus post-transcriptional regulatory element

Chronic hepatitis B virus (HBV) infections may lead to severe diseases like liver cirrhosis or hepatocellular carcinoma (HCC). The HBV post-transcriptional regulatory element (HPRE) facilitates the nuclear export of unspliced viral mRNAs, contains a splicing regulatory element and resides in the 3′-region of all viral transcripts. The HPRE consists of three sub-elements α (nucleotides 1151–1346), β1 (nucleotides 1347–1457) and β2 (nucleotides 1458–1582), which confer together full export competence. Here, we present the NMR solution structure (pdb 2JYM) of the stem-loop α (SLα, nucleotides 1292–1321) located in the sub-element α. The SLα contains a CAGGC pentaloop highly conserved in hepatoviruses, which essentially adopts a CUNG-like tetraloop conformation. Furthermore, the SLα harbours a single bulged G residue flanked by A-helical regions. The structure is highly suggestive of serving two functions in the context of export of unspliced viral RNA: binding sterile alpha motif (SAM-) domain containing proteins and/or preventing the utilization of a 3′-splice site contained within SLα.

Cellular Protein TIA-1 Regulates the Expression of HBV Surface Antigen by Binding the HBV Posttranscriptional Regulatory Element

Objective: To identify T-cell intracellular antigen 1 (TIA-1) interacting with the posttranscriptional regulatory element (PRE) of hepatitis B virus (HBV), and reveal TIA-1 function on HBV gene expression. Methods: In order to demonstrate TIA-1 binding to PRE, electrophoretic mobility shift and RNA and protein pull-down assays were used. The pDM138-PRE reporter system, HepG2.2.15 cell line and HBs-PRE transient expression cells were adopted to identify TIA-1 function in the HBV life cycle. Results: TIA-1 could directly bind to PRE RNA. The functional binding region of TIA-1 was identified. Functional analysis indicated that TIA-1 could dose-dependently inhibit PRE function in pDM138-PRE system. Down-expression of HBs antigen induced by TIA-1 was detected both in the HepG2.2.15 cell line and in HBs-PRE transient expression cells. Conclusion: These data demonstrated that TIA-1 inhibits HBs expression by interacting with PRE.

Splicing-independent expression of the herpes simplex virus type 1 thymidine kinase gene is mediated by three cis-acting RNA subelements.

Herpes simplex virus genes are predominantly intronless. We identified cis-acting elements in the intronless herpes simplex virus type 1 thymidine kinase (TK) gene that facilitate intron-independent gene expression. TK sequences functionally replaced the hepatitis B virus (HBV) posttranscriptional regulatory element (PRE) by inducing the expression of the intronless HBV surface message. TK also activated the pDM138 assay by inducing the cytoplasmic accumulation of intron-containing RNA. Multiple cis-acting RNA sequences, or subelements, that induce cytoplasmic localization of unspliced RNA were mapped within the TK gene. The presence of multiple RNA subelements within the TK gene is reminiscent of the multiple subelements in the HBV PRE required for the cytoplasmic accumulation of intronless HBV RNAs. Similar to HBV PRE subelements, duplication of a single TK subelement resulted in greater-than-additive increases in activity. A reporter chimera containing a single TK subelement juxtaposed to an HBV PRE subelement demonstrated a commensurate increase in activity. These results suggest that viral intronless genes utilize a similar strategy for intron-independent gene expression that requires multiple cis-acting RNA signals. Furthermore, like HBV PRE-containing RNA, TK cytoplasmic localization is not sensitive to leptomycin B, a drug that inhibits the export of proteins containing nuclear export signals. From this, we conclude that proteins that bind TK and facilitate its cytoplasmic accumulation do not travel through a CRM1-dependent RNA transport pathway.

Leptomycin B inhibits equine infectious anemia virus Rev and feline immunodeficiency virus rev function but not the function of the hepatitis B virus posttranscriptional regulatory element.

Human immunodeficiency virus type 1 Rev export depends upon the presence of the nuclear export signal (NES), a leucine-rich stretch of hydrophobic amino acids. Recently, the nuclear NES-binding receptor has been identified as CRM1 or exportin 1. Rev export has been shown to be CRM1 dependent. The function of the atypical NES-containing Rev-like proteins of equine infectious anemia virus (EIAV) and feline immunodeficiency virus (FIV) is inhibited by leptomycin B, a drug that specifically blocks NES-CRM1 interactions. These data suggest that the function of atypical NES-containing proteins is CRM1 dependent. In contrast to the inhibition of EIAV Rev and FIV Rev, the cytoplasmic accumulation of hepatitis B virus (HBV) posttranscriptional regulatory element (PRE)-containing and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-containing RNAs is not inhibited by leptomycin B treatment. We conclude that the HBV PRE, like the MPMV CTE, functions independently of an NES receptor-exportin 1 interaction.

Woodchuck hepatitis virus contains a tripartite posttranscriptional regulatory element.

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.

The hepatitis B virus posttranscriptional regulatory element is composed of two subelements.

The RNAs of the hepatitis B virus (HBV) contain a cis-acting regulatory element which facilitates the cytoplasmic localization of unspliced transcripts (J. Huang and T. J. Liang, Mol. Cell. Biol. 13:7476-7486, 1993, and Z. M. Huang and T. S. Yen, J. Virl. 68:3193-3199, 1994). Such localization is presumed to be mediated by cellular factors which interact with the element. The HBV posttranscriptional regulatory element (HBVPRE) can efficiently activate an RNA export reporter system in an orientation-dependent and position-independent manner. Deletion analysis reveals that the HBVPRE consists of two subelements which function synergistically. A synergistic effect was also observed when the 5' (PREalpha) or 3' (PREbeta) subelements were duplicated. The bipartite structure of the HBVPRE is reminiscent of reports that the high-affinity binding sites of the Rev-like proteins must be duplicated to function efficiently (M. Grone, E. Hoffmann, S. Berchtold, B.R. Cullen, and R. Grassmann, Virology 204:144-152, 1994; X. Huang, T.J. Hope, B.L. Bond, D. McDonald, K. Grahl, and T. G. Parslow, J. Virol. 65:2131-2134, 1991; and D. McDonald, T. J. Hope, and T. G. Parslow, J. Virol. 66:7232-7238, 1992).