scholarly journals Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production

2020 ◽  
Vol 16 (11) ◽  
pp. e1008593
Author(s):  
Hélène Chabrolles ◽  
Héloïse Auclair ◽  
Serena Vegna ◽  
Thomas Lahlali ◽  
Caroline Pons ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Core Protein ◽  
Restriction Factor ◽  
Dependent Manner ◽  
New Host ◽  
Functional Studies ◽  
B Virus

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.

scholarly journals Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production

2020 ◽  
Author(s):  
Hélène Chabrolles ◽  
Héloïse Auclair ◽  
Serena Vegna ◽  
Thomas Lahlali ◽  
Caroline Pons ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Core Protein ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
B Virus ◽  
Regulatory Functions

AbstractDespite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.Author SummaryChronic infection with Hepatitis B virus (HBV) affects more than 250 millions of people world-wide and is a major global cause of liver cancer. Current treatments lead to a significant reduction of viremia in patients. However, viral clearance is rarely obtained and the persistence of the HBV genome in the hepatocyte’s nucleus generates a stable source of viral RNAs and subsequently proteins which play important roles in immune escape mechanisms and liver disease progression. Therapies aiming at efficiently and durably eliminating viral gene expression are still required. In this study, we identified the nuclear partners of the HBV Core protein (HBc) to understand how this structural protein, responsible for capsid assembly in the cytoplasm, could also regulate viral gene expression. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs). One of these RBPs, SRSF10, was demonstrated to restrict HBV RNA levels and a drug, able to alter its phosphorylation, behaved as an antiviral compound capable of reducing viral gene expression. Altogether, this study sheds new light novel regulatory functions of HBc and provides information relevant for the development of antiviral strategies aiming at preventing viral gene expression.

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 SOX2 Represses Hepatitis B Virus Replication by Binding to the Viral EnhII/Cp and Inhibiting the Promoter Activation

Viruses ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 273
Author(s):  
Hua Yang ◽  
Jiayin Mo ◽  
Qi Xiang ◽  
Peiyi Zhao ◽  
Yunting Song ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Protein ◽  
Hepatitis B Surface Antigen ◽  
High Mobility ◽  
Promoter Activation ◽  
New Host ◽  
Rna Transcription ◽  
Hbv Replication ◽  
B Virus

Hepatitis B virus (HBV) replication is controlled by four promoters (preS1, preS2, Cp, and Xp) and two enhancers (EnhI and EnhII). EnhII stimulates Cp activity to regulate the transcriptions of precore, core, polymerase, and pregenomic RNAs, and therefore, EnhII/Cp is essential for the regulation of HBV replication. This study revealed a distinct mechanism underlying the suppression of EnhII/Cp activation and HBV replication. On the one hand, the sex determining region Y box2 (SOX2), a transcription factor, is induced by HBV. On the other hand, SOX2, in turn, represses the expression levels of HBV RNAs, HBV core-associated DNA, hepatitis B surface antigen (HBsAg), and hepatitis B e antigen (HBeAg), thereby playing an inhibitory role during HBV replication. Further studies indicated that SOX2 bound to the EnhII/Cp DNA and repressed the promoter activation. With the deletion of the high mobility group (HMG) domain, SOX2 loses the ability to repress EnhII/Cp activation, viral RNA transcription, HBV core-associated DNA replication, HBsAg and HBeAg production, as well as fails to enter the nucleus, demonstrating that the HMG domain is required for the SOX2-mediated repression of HBV replication. Moreover, SOX2 represses HBsAg and HBeAg secretion in BALB/c mice sera, and attenuates HBV 3.5 kb RNA transcription and hepatitis B virus core protein (HBc) production in the liver tissues, demonstrating that SOX2 suppresses HBV replication in mice. Furthermore, the results revealed that the HMG domain was required for SOX2-mediated repression of HBV replication in the mice. Taken together, the above facts indicate that SOX2 acts as a new host restriction factor to repress HBV replication by binding to the viral EnhII/Cp and inhibiting the promoter activation through the HMG domain.

scholarly journals Negative regulation of hepatitis B virus replication by cellular Hsp40/DnaJ proteins through destabilization of viral core and X proteins

2006 ◽  
Vol 87 (7) ◽  
pp. 1883-1891 ◽  
Author(s):  
Sook-Young Sohn ◽  
Sun-Bum Kim ◽  
Joon Kim ◽  
Byung-Yoon Ahn
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Virus Replication ◽  
Rna Binding ◽  
Core Protein ◽  
Ectopic Expression ◽  
Viral Core ◽  
Amino Terminal ◽  
B Virus ◽  
Carboxyl Terminal

The hepatitis B virus core protein consists of an amino-terminal capsid-assembly domain and a carboxyl-terminal RNA-binding domain. By using the yeast two-hybrid system, two Hsp40/DnaJ chaperone-family proteins, Hdj1 and hTid1, that interact with the carboxyl-terminal region (aa 94–185) of the core protein were identified. Hdj1 is the prototype member of the family and hTid1 is the human homologue of the Drosophila tumour-suppressor protein Tid56. Binding of the viral core protein with the Hsp40 proteins was confirmed by affinity chromatography and immunoprecipitation of transiently expressed proteins. Moreover, in a sucrose gradient, the precursor form of hTid1 co-sedimented with capsid-like particles composed of the full-length core protein. Unlike the general perception of the role of the cellular chaperone proteins in assisting viral protein folding and thus enhancing virus replication, ectopic expression of Hdj1 and hTid1 suppressed replication of HBV in transfected human hepatoma cells. Conversely, RNA interference-mediated knock-down of hTid1 resulted in increased HBV replication. It was found that both Hsp40 proteins specifically accelerated degradation of the viral core and HBx proteins. Our results suggest that the cellular chaperones, through destabilization of viral proteins, exert inhibitory functions on virus replication and hence may play suppressive roles in hepatocellular carcinoma.

scholarly journals Full-Length Hepatitis B Virus Core Protein Packages Viral and Heterologous RNA with Similarly High Levels of Cooperativity

2010 ◽  
Vol 84 (14) ◽  
pp. 7174-7184 ◽  
Author(s):  
J. Zachary Porterfield ◽  
Mary Savari Dhason ◽  
Daniel D. Loeb ◽  
Michael Nassal ◽  
Stephen J. Stray ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Rna Binding ◽  
Core Protein ◽  
Viral Polymerase ◽  
Critical Feature ◽  
Protein Dimer ◽  
B Virus

ABSTRACT A critical feature of a viral life cycle is the ability to selectively package the viral genome. In vivo, phosphorylated hepatitis B virus (HBV) core protein specifically encapsidates a complex of pregenomic RNA (pgRNA) and viral polymerase; it has been suggested that packaging is specific for the complex. Here, we test the hypothesis that core protein has intrinsic specificity for pgRNA, independent of the polymerase. For these studies, we also evaluated the effect of core protein phosphorylation on assembly and RNA binding, using phosphorylated core protein and a phosphorylation mimic in which S155, S162, and S170 were mutated to glutamic acid. We have developed an in vitro system where capsids are disassembled and assembly-active core protein dimer is purified. With this protein, we have reassembled empty capsids and RNA-filled capsids. We found that core protein dimer bound and encapsidated both the HBV pregenomic RNA and heterologous RNA with high levels of cooperativity, irrespective of phosphorylation. In direct competition assays, no specificity for pregenomic RNA was observed. This suggests that another factor, such as the viral polymerase, is required for specific packaging. These results also beg the question of what prevents HBV core protein from assembling on nonviral RNA, preserving the protein for virus production.

scholarly journals Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure

2018 ◽  
Vol 14 (12) ◽  
pp. e1007488 ◽  
Author(s):  
Julia Heger-Stevic ◽  
Peter Zimmermann ◽  
Lauriane Lecoq ◽  
Bettina Böttcher ◽  
Michael Nassal
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Protein Phosphorylation ◽  
Rna Binding ◽  
Core Protein ◽  
Virus Core ◽  
B Virus ◽  
Target Sites

scholarly journals Inhibition of Hepatitis B Virus Gene Expression and Replication by Helioxanthin and its Derivative

2005 ◽  
Vol 16 (3) ◽  
pp. 193-201 ◽  
Author(s):  
Ying Li ◽  
Lei Fu ◽  
Hosup Yeo ◽  
Ju-Liang Zhu ◽  
Chen-Kung Chou ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Protein Expression ◽  
Core Protein ◽  
Hbv Dna ◽  
Interferon Α ◽  
Dependent Manner ◽  
Therapeutic Drugs ◽  
Chronic Hepatitis B Virus ◽  
B Virus

Chronic hepatitis B virus (HBV) infection continues to be an important worldwide cause of morbidity and mortality. All the currently approved therapeutic drugs have their limitations: interferon-α (IFN-α) has limited efficacy and a high incidence of adverse effects; nucleoside analogues are very efficient HBV DNA inhibitors, but resistance occurs eventually. Therefore, it is important to develop new non-nucleoside/nucleotide agents with different modes of action that can be used for antiviral combination therapy. Here, we report on a novel class of compounds, helioxanthin and its derivative 5-4-2, which had potent anti-HBV activities in HepG2.2.15 cells, with the EC50s of 1 and 0.08 μM, respectively. The lamivudine-resistant HBV, L526M/M550V double mutant strain, was also sensitive to helioxanthin and 5-4-2. This class of compounds not only inhibited HBV DNA, but also decreased HBV mRNA and HBV protein expression. The EC50 of HBV DNA inhibition was consistent with the EC50 of HBV 3.5 Kb transcript inhibition, which was 1 and 0.09 μM for helioxanthin and 5-4-2 respectively. Western blot analysis of cell lysate from HepG2.2.15 cells showed that the core protein expression decreased in a dose-dependent manner after drug treatment. In conclusion, helioxanthin and 5-4-2 are potentially unique new anti-HBV agents, which possess a different mechanism of action from existing therapeutic drugs. Both compounds inhibited HBV RNA and protein expression in addition to inhibiting HBV DNA.

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