scholarly journals Comparative Analysis of Hepatitis B Virus Polymerase Sequences Required for Viral RNA Binding, RNA Packaging, and Protein Priming

2013 ◽  
Vol 88 (3) ◽  
pp. 1564-1572 ◽  
Author(s):  
S. A. Jones ◽  
D. N. Clark ◽  
F. Cao ◽  
J. E. Tavis ◽  
J. Hu
Keyword(s):  
Comparative Analysis ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Rna Binding ◽  
Viral Rna ◽  
Rna Packaging ◽  
B Virus ◽  
Protein Priming

scholarly journals Hepatitis B Virus RNA-Binding Proteins Associated with Cytokine-Induced Clearance of Viral RNA from the Liver of Transgenic Mice

1999 ◽  
Vol 73 (1) ◽  
pp. 474-481 ◽  
Author(s):  
Tilman Heise ◽  
Luca G. Guidotti ◽  
Victoria J. Cavanaugh ◽  
Francis V. Chisari
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Transgenic Mice ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Viral Rna ◽  
Cytokine Activation ◽  
Tnf Α ◽  
B Virus ◽  
Ifn Γ

ABSTRACT Hepatitis B virus (HBV) gene expression is downregulated in the liver of HBV transgenic mice by a posttranscriptional mechanism that is triggered by the local production of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) during intrahepatic inflammation (hepatitis). The molecular basis for this antiviral effect is unknown. In this study, we identified three HBV RNA-binding liver nuclear proteins (p45, p39, and p26) the relative abundance of which correlates with the abundance of HBV RNA in response to the induction of IFN-γ and TNF-α. All three proteins bind to a 91-bp element located at the 5′ end of a previously defined posttranscriptional regulatory element that is thought to mediate the nuclear export of HBV RNA. The presence of p45 correlates directly with the presence of HBV RNA, being detectable under baseline conditions when the viral RNA is abundant and undetectable when the viral RNA disappears in response to IFN-γ and TNF-α. In contrast, p26 is inversely related to HBV RNA, being detectable only when the viral RNA disappears following cytokine activation. Finally, p39 is constitutively expressed, and its abundance and mobility appear to be slightly increased by cytokine activation. These results suggest a model in which hepatocellular HBV RNA content might be controlled by the stabilizing and/or destabilizing influences of these RNA-binding proteins whose activity is regulated by cytokine-induced signaling pathways.

scholarly journals Capsid Phosphorylation State and Hepadnavirus Virion Secretion

2017 ◽  
Vol 91 (9) ◽  
Author(s):  
Xiaojun Ning ◽  
Suresh H. Basagoudanavar ◽  
Kuancheng Liu ◽  
Laurie Luckenbaugh ◽  
Duoqian Wei ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Synthesis ◽  
Potential Role ◽  
Viral Rna ◽  
Rna Packaging ◽  
Viral Dna ◽  
Phosphorylation State ◽  
B Virus

ABSTRACT The C-terminal domain (CTD) of hepadnavirus core protein is involved in multiple steps of viral replication. In particular, the CTD is initially phosphorylated at multiple sites to facilitate viral RNA packaging into immature nucleocapsids (NCs) and the early stage of viral DNA synthesis. For the avian hepadnavirus duck hepatitis B virus (DHBV), CTD is dephosphorylated subsequently to facilitate the late stage of viral DNA synthesis and to stabilize NCs containing mature viral DNA. The role of CTD phosphorylation in virion secretion, if any, has remained unclear. Here, the CTD from the human hepatitis B virus (HBV) was found to be dephosphorylated in association with NC maturation and secretion of DNA-containing virions, as in DHBV. In contrast, the CTD in empty HBV virions (i.e., enveloped capsids with no RNA or DNA) was found to be phosphorylated. The potential role of CTD dephosphorylation in virion secretion was analyzed through mutagenesis. For secretion of empty HBV virions, which is independent of either viral RNA packaging or DNA synthesis, multiple substitutions in the CTD to mimic either phosphorylation or dephosphorylation showed little detrimental effect. Similarly, phospho-mimetic substitutions in the DHBV CTD did not block the secretion of DNA-containing virions. These results indicate that CTD dephosphorylation, though associated with NC maturation in both HBV and DHBV, is not essential for the subsequent NC-envelope interaction to secrete DNA-containing virions, and the CTD state of phosphorylation also does not play an essential role in the interaction between empty capsids and the envelope for secretion of empty virions. IMPORTANCE The phosphorylation state of the C-terminal domain (CTD) of hepatitis B virus (HBV) core or capsid protein is highly dynamic and plays multiple roles in the viral life cycle. To study the potential role of the state of phosphorylation of CTD in virion secretion, we have analyzed the CTD phosphorylation state in complete (containing the genomic DNA) versus empty (genome-free) HBV virions. Whereas CTD is unphosphorylated in complete virions, it is phosphorylated in empty virions. Mutational analyses indicate that neither phosphorylation nor dephosphorylation of CTD is required for virion secretion. These results demonstrate that while CTD dephosphorylation is associated with HBV DNA synthesis, the CTD state of phosphorylation may not regulate virion secretion.

scholarly journals Hepatitis B virus polymerase restricts LINE-1 retrotransposition

2021 ◽  
Author(s):  
Yasuo Ariumi
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Rna Binding ◽  
Rnase H ◽  
Ribonuclease H ◽  
Independent Manner ◽  
Cytoplasmic Rna ◽  
B Virus ◽  
Long Interspersed Element ◽  
L1 Retrotransposon

Long interspersed element-1 (LINE-1, L1) retrotransposon composes about 17% of the human genome. However, genetic and biochemical interactions between L1 and hepatitis B virus (HBV) remain poorly understood. In this study, we found that HBV restricts L1 mobility without inhibiting the L1 promoter activity. Notably, HBV polymerase (Pol) strongly inhibited L1 retrotransposition in a reverse transcriptase (RT)-independent manner. Indeed, the ribonuclease H (RNase H) domain was essential for inhibition of L1 retrotransposition. L1 ORF1p RNA-binding protein predominantly localized into cytoplasmic RNA granule termed P-body. However, HBV Pol sequestered L1 ORF1p from P-body and colocalized with L1 ORF1p in cytoplasm, when both proteins were co-expressed. Altogether, HBV Pol seems to restrict L1 mobility through a sequestration of L1 ORF1p from P-body. Thus, these results suggest a novel function or activity of HBV Pol in regulation of L1 retrotransposition.

Inhibition of Duck Hepatitis B Virus Replication by 9-(2-Phosphonylmethoxyethyl)adenine, an Acyclic Phosphonate Nucleoside Analogue

1998 ◽  
Vol 42 (12) ◽  
pp. 3130-3135 ◽  
Author(s):  
A. J. Nicoll ◽  
D. L. Colledge ◽  
J. J. Toole ◽  
P. W. Angus ◽  
R. A. Smallwood ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Nucleoside Analogue ◽  
Viral Rna ◽  
Northern Hybridization ◽  
Duck Hepatitis B Virus ◽  
Protein Levels ◽  
Duck Hepatitis ◽  
Chronic Hepatitis B Virus ◽  
B Virus

ABSTRACT The use of regimens that use nucleoside analogues for the treatment of chronic hepatitis B virus infection is often limited because of their high relapse rates. This is thought to be due to the persistence of virus in nonhepatocyte reservoirs and/or the viral covalently closed circular (CCC) DNA species in the nucleus of infected hepatocytes. We have evaluated the novel nucleoside analogue 9-(2-phosphonylmethoxyethyl)adenine (PMEA) in the duck model of hepatitis B. Eight Pekin-Aylesbury ducks congenitally infected with the duck hepatitis B virus (DHBV) were treated with PMEA at a dosage of 15 mg/kg of body weight/day via the intraperitoneal route for 4 weeks. At the end of the treatment period, four animals were killed and the remainder were monitored for a further 4-week drug-free period before analysis. The results were compared with those for eight age-matched, untreated controls. The levels of viremia, the total intrahepatic DHBV load, and CCC DNA, viral RNA, and protein levels were measured by Southern hybridization, Northern hybridization, and immunoblotting of the appropriate specimen, respectively. Viral proteins and DNA were also measured by immunohistochemistry (IHC) and in situ hybridization (ISH) of sections of liver and pancreatic tissue. PMEA treatment reduced the viremia to undetectable levels, while the total viral DNA load in the liver was reduced by 95% compared to the control level. Viral RNA and protein levels decreased by approximately 30%. ISH and IHC confirmed the PMEA-related intrahepatic changes and established that the amount of virus in bile duct epithelial cells (BDEC) was reduced by 70% during therapy. During the follow-up period all parameters of active virological replication returned to those for the age-matched controls. PMEA had no significant effect upon the number of virus-infected islet or acinar cells in the pancreas. PMEA at a dosage of 15 mg/kg/day has potent activity against DHBV found within hepatocytes and BDEC and inhibits DHBV replication in BDEC.

scholarly journals Hepatitis B Virus X Upregulates HuR Protein Level to Stabilize HER2 Expression in Hepatocellular Carcinoma Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Chao-Ming Hung ◽  
Wei-Chien Huang ◽  
Hsiao-Lin Pan ◽  
Pei-Hsuan Chien ◽  
Chih-Wen Lin ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Protein Level ◽  
Rna Binding ◽  
Signal Pathways ◽  
Her2 Expression ◽  
Migration Ability ◽  
Mrna Stabilization ◽  
B Virus

Hepatitis B virus- (HBV-) associated hepatocellular carcinoma (HCC) is the most common type of liver cancer. However, the underlying mechanism of HCC tumorigenesis is very complicated and HBV-encoded X protein (HBx) has been reported to play the most important role in this process. Activation of downstream signal pathways of epidermal growth factor receptor (EGFR) family is known to mediate HBx-dependent HCC tumor progression. Interestingly, HER2 (also known as ErbB2/Neu/EGFR2) is frequently overexpressed in HBx-expressing HCC patients and is associated with their poor prognosis. However, it remains unclear whether and how HBx regulates HER2 expression. In this study, our data showed that HBx expression increased HER2 protein level via enhancing its mRNA stability. The induction of RNA-binding protein HuR expression by HBx mediated the HER2 mRNA stabilization. Finally, the upregulated HER2 expression promoted the migration ability of HBx-expressing HCC cells. These findings deciphered the molecular mechanism of HBx-mediated HER2 upregulation in HBV-associated HCC.

scholarly journals In Vitro Reconstitution of a Functional Duck Hepatitis B Virus Reverse Transcriptase: Posttranslational Activation by Hsp90

2000 ◽  
Vol 74 (24) ◽  
pp. 11447-11455 ◽  
Author(s):  
Jianming Hu ◽  
Dana Anselmo
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcriptase ◽  
Host Factors ◽  
Duck Hepatitis B Virus ◽  
Duck Hepatitis ◽  
B Virus ◽  
Protein Priming ◽  
Hsp90 Function

ABSTRACT Reverse transcription in hepatitis B viruses is initiated through a unique protein priming mechanism whereby the viral reverse transcriptase (RT) first assembles into a ribonucleoprotein (RNP) complex with its RNA template and then initiates DNA synthesis de novo using the RT itself as a protein primer. RNP formation and protein priming require the assistance of host cell factors, including the molecular chaperone heat shock protein 90 (Hsp90). To better understand the mechanism of RT activation by Hsp90, we have now mapped the minimal RT sequences of the duck hepatitis B virus that are required for chaperone binding, RNP formation, and protein priming. Furthermore, we have reconstituted in vitro both RNP formation and protein priming using purified RT proteins and host factors. Our results show that (i) Hsp90 recognizes two independent domains of the RT, both of which are necessary for RNP formation and protein priming; (ii) Hsp90 function is required not only to establish, but also to maintain, the RT in a state competent for RNA binding; and (iii) Hsp90 is not required during RT synthesis and can activate the RT posttranslationally. Based on these findings, we propose a model for Hsp90 function whereby the chaperone acts as an active interdomain bridge to bring the two RT domains into a poised but labile conformation competent for RNP formation. It is anticipated that the reconstitution system established here will facilitate the isolation of additional host factors required for RT functions and further elucidation of the mechanisms of RT activation.

scholarly journals Characterization of Nuclear RNases That Cleave Hepatitis B Virus RNA near the La Protein Binding Site

2001 ◽  
Vol 75 (15) ◽  
pp. 6874-6883 ◽  
Author(s):  
Tilman Heise ◽  
Luca G. Guidotti ◽  
Francis V. Chisari
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Rna Binding ◽  
Regulatory Element ◽  
Murine Cytomegalovirus ◽  
Mcmv Infection ◽  
Stem Loop ◽  
La Protein ◽  
Nuclear Extracts ◽  
B Virus

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.

scholarly journals Mapping of Functional Subdomains in the Terminal Protein Domain of Hepatitis B Virus Polymerase

2016 ◽  
Vol 91 (3) ◽  
Author(s):  
Daniel N. Clark ◽  
John M. Flanagan ◽  
Jianming Hu
Keyword(s):  
Hepatitis B Virus ◽  
High Resolution ◽  
Secondary Structure ◽  
Hepatitis B ◽  
Reverse Transcription ◽  
Viral Rna ◽  
Terminal Protein ◽  
B Virus ◽  
High Resolution Structure ◽  
Resolution Structure

ABSTRACTHepatitis B virus (HBV) encodes a multifunction reverse transcriptase or polymerase (P), which is composed of several domains. The terminal protein (TP) domain is unique to HBV and related hepadnaviruses and is required for specifically binding to the viral pregenomic RNA (pgRNA). Subsequently, the TP domain is necessary for pgRNA packaging into viral nucleocapsids and the initiation of viral reverse transcription for conversion of the pgRNA to viral DNA. Uniquely, the HBV P protein initiates reverse transcription via a protein priming mechanism using the TP domain as a primer. No structural homologs or high-resolution structure exists for the TP domain. Secondary structure prediction identified three disordered loops in TP with highly conserved sequences. A meta-analysis of mutagenesis studies indicated these predicted loops are almost exclusively where functionally important residues are located. Newly constructed TP mutations revealed a priming loop in TP which plays a specific role in protein-primed DNA synthesis beyond simply harboring the site of priming. Substitutions of potential sites of phosphorylation surrounding the priming site demonstrated that these residues are involved in interactions critical for priming but are unlikely to be phosphorylated during viral replication. Furthermore, the first 13 and 66 TP residues were shown to be dispensable for protein priming and pgRNA binding, respectively. Combining current and previous mutagenesis work with sequence analysis has increased our understanding of TP structure and functions by mapping specific functions to distinct predicted secondary structures and will facilitate antiviral targeting of this unique domain.IMPORTANCEHBV is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. One important feature of this virus is its polymerase, the enzyme used to create the DNA genome from a specific viral RNA by reverse transcription. One region of this polymerase, the TP domain, is required for association with the viral RNA and production of the DNA genome. Targeting the TP domain for antiviral development is difficult due to the lack of homology to other proteins and high-resolution structure. This study mapped the TP functions according to predicted secondary structure, where it folds into alpha helices or unstructured loops. Three predicted loops were found to be the most important regions functionally and the most conserved evolutionarily. Identification of these functional subdomains in TP will facilitate its targeting for antiviral development.

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