Inhibition of Viral Replication Reduces Transcriptionally Active Distinct Hepatitis B Virus Integrations with Implications on Host Gene Dysregulation

ABSTRACT Control of viral replication is a major therapeutic goal to reduce morbidity and mortality from chronic hepatitis B virus (HBV) infection. Recently, methylation has been identified as a novel host defense mechanism, and methylation of viral DNA leads to downregulation of HBV gene expression. To better understand the mechanisms of HBV methylation, cell lines were exposed to HBV using a model system that mimics natural infection and the expression of host DNA methyltransferase genes (DNMTs) was measured. DNMT1, DNMT2, and DNMT3 were all significantly upregulated in response to HBV. DNMT3 was further studied because of its known role in the de novo methylation of DNA. Cotransfection experiments with full-length HBV and DNMT3 led to the downregulation of viral protein and pregenomic RNA production. To investigate whether the upregulation of DNMTs could also have an effect on the methylation of host DNA, cell lines were exposed to HBV in two independent model systems, one that mimics natural infection and a second model with temporary transfection. Host DNA methylation was measured by DNA microarray analysis. Increased methylation of host CpG islands was detected in both experimental systems. Two CpG islands, corresponding to genes SUFU and TIRAP, were selected, and the downregulation of these genes in hepatocellular carcinomas was confirmed. In conclusion, hepatocytes respond to HBV infection by upregulating DNMTs. The DNMTs methylate viral DNA, leading to decreased viral gene expression and decreased viral replication. However, virus-induced overexpression of DNMTs also leads to methylation of host CpG islands.

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Covalently Closed Circular DNA Is the Predominant Form of Duck Hepatitis B Virus DNA That Persists following Transient Infection

Journal of Virology ◽

10.1128/jvi.79.19.12242-12252.2005 ◽

2005 ◽

Vol 79 (19) ◽

pp. 12242-12252 ◽

Cited By ~ 31

Author(s):

Marc F. Le Mire ◽

Darren S. Miller ◽

Wendy K. Foster ◽

Christopher J. Burrell ◽

Allison R. Jilbert

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Viral Replication ◽

Mononuclear Cells ◽

Blot Hybridization ◽

Immunosuppressive Treatment ◽

Hbv Dna ◽

Duck Hepatitis B Virus ◽

Duck Hepatitis ◽

B Virus

ABSTRACT Residual hepatitis B virus (HBV) DNA can be detected in serum and liver after apparent recovery from transient infection. However, it is not known if this residual HBV DNA represents ongoing viral replication and antigen expression. In the current study, ducks inoculated with duck hepatitis B virus (DHBV) were monitored for residual DHBV DNA following recovery from transient infection until 9 months postinoculation (p.i.). Resolution of DHBV infection occurred in 13 out of 15 ducks by 1-month p.i., defined as clearance of DHBV surface antigen-positive hepatocytes from the liver and development of anti-DHBV surface antibodies. At 9 months p.i., residual DHBV DNA was detected using nested PCR in 10/11 liver, 7/11 spleen, 2/11 kidney, 1/11 heart, and 1/11 adrenal samples. Residual DHBV DNA was not detected in serum or peripheral blood mononuclear cells. Within the liver, levels of residual DHBV DNA were 0.0024 to 0.016 copies per cell, 40 to 80% of which were identified as covalently closed circular viral DNA by quantitative PCR assay. This result, which was confirmed by Southern blot hybridization, is consistent with suppressed viral replication or inactive infection. Samples of liver and spleen cells from recovered animals did not transmit DHBV infection when inoculated into 1- to 2-day-old ducklings, and immunosuppressive treatment of ducks with cyclosporine and dexamethasone for 4 weeks did not alter levels of residual DHBV DNA in the liver. These findings further characterize a second form of hepadnavirus persistence in a suppressed or inactive state, quite distinct from the classical chronic carrier state.

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Discovery and Selection of Hepatitis B Virus-Derived T Cell Epitopes for Global Immunotherapy Based on Viral Indispensability, Conservation, and HLA-Binding Strength

Journal of Virology ◽

10.1128/jvi.01663-19 ◽

2019 ◽

Vol 94 (7) ◽

Cited By ~ 1

Author(s):

Monique T. A. de Beijer ◽

Diahann T. S. L. Jansen ◽

Yingying Dou ◽

Wim J. E. van Esch ◽

Juk Yee Mok ◽

...

Keyword(s):

Hepatitis B Virus ◽

T Cell ◽

Hepatitis B ◽

Viral Replication ◽

Immune Escape ◽

The Novel ◽

T Cell Epitopes ◽

Infected Population ◽

B Virus ◽

Hla Binding

ABSTRACT Immunotherapy represents an attractive option for the treatment of chronic hepatitis B virus (HBV) infection. The HBV proteins polymerase (Pol) and HBx are of special interest for antigen-specific immunotherapy because they are essential for viral replication and have been associated with viral control (Pol) or are still expressed upon viral DNA integration (HBx). Here, we scored all currently described HBx- and Pol-derived epitope sequences for viral indispensability and conservation across all HBV genotypes. This yielded 7 HBx-derived and 26 Pol-derived reported epitopes with functional association and high conservation. We subsequently predicted novel HLA-binding peptides for 6 HLA supertypes prevalent in HBV-infected patients. Potential epitopes expected to be the least prone to immune escape were subjected to a state-of-the-art in vitro assay to validate their HLA-binding capacity. Using this method, a total of 13 HLA binders derived from HBx and 33 binders from Pol were identified across HLA types. Subsequently, we demonstrated interferon gamma (IFN-γ) production in response to 5 of the novel HBx-derived binders and 17 of the novel Pol-derived binders. In addition, we validated several infrequently described epitopes. Collectively, these results specify a set of highly potent T cell epitopes that represent a valuable resource for future HBV immunotherapy design. IMPORTANCE Multiple HBV-derived T cell epitopes have been reported, which can be useful in a therapeutic vaccination strategy. However, these epitopes are largely restricted to HLA-A*02, which is not dominantly expressed in populations with high HBV prevalence. Thus, current epitopes are falling short in the development of a global immunotherapeutic approach. Therefore, we aimed to identify novel epitopes for 6 HLA supertypes most prevalent in the infected population. Moreover, established epitopes might not all be equally effective as they can be subject to different levels of immune escape. It is therefore important to identify targets that are crucial in viral replication and conserved in the majority of the infected population. Here, we applied a stringent selection procedure to compose a combined overview of existing and novel HBV-derived T cell epitopes most promising for viral eradication. This set of T cell epitopes now lays the basis for the development of globally effective HBV antigen-specific immunotherapies.

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Host Gene SEL1L Involved in Endoplasmic Reticulum-Associated Degradation Pathway Could Inhibit Hepatitis B Virus at RNA, DNA, and Protein Levels

Frontiers in Microbiology ◽

10.3389/fmicb.2019.02869 ◽

2019 ◽

Vol 10 ◽

Cited By ~ 2

Author(s):

Jinyu Wang ◽

Jing Li ◽

Jingwen Wu ◽

Minhui Dong ◽

Zhongliang Shen ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Degradation Pathway ◽

Host Gene ◽

Protein Levels ◽

Endoplasmic Reticulum Associated Degradation ◽

B Virus

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Activation of Focal Adhesion Kinase by Hepatitis B Virus HBx Protein: Multiple Functions in Viral Replication

Journal of Virology ◽

10.1128/jvi.80.9.4406-4414.2006 ◽

2006 ◽

Vol 80 (9) ◽

pp. 4406-4414 ◽

Cited By ~ 71

Author(s):

Michael J. Bouchard ◽

Lihua Wang ◽

Robert J. Schneider

Keyword(s):

Signal Transduction ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Viral Replication ◽

Calcium Signaling ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Cellular Transformation ◽

B Virus ◽

Stimulation Of

ABSTRACT The hepatitis B virus (HBV) X protein (HBx) is a multifunctional regulator of cellular signal transduction and transcription pathways and has a critical role in HBV replication. Much of the cytoplasmic signal transduction activity associated with HBx expression and its stimulation of viral replication is attributable to HBx-induced activation of calcium signaling pathways involving Pyk2 and Src tyrosine kinases. To further characterize upstream signal transduction pathways that are required for HBx activity, including activation of Src and mitogen-activated protein kinase (MAPK) cascades, we determined whether focal adhesion kinase (FAK), a known regulator of Src family kinases and the other member of the Pyk2/FAK kinase family, is activated by HBx. We report that HBx activates FAK and that FAK activation is important for multiple HBx functions. Dominant inhibiting forms of FAK blocked HBx activation of Src kinases and downstream signal transduction, HBx stimulation of NF-κB and AP-1-dependent transcription, and HBV DNA replication. We also demonstrate that HBx-induced activation of FAK is dependent on cellular calcium signaling, which is modulated by HBx. Moreover, prolonged expression of HBx increases both FAK activity and its level of expression. FAK activation may play a role in cellular transformation and cancer progression. HBx stimulation of FAK activity and abundance may also be relevant as a potential cofactor in HBV-associated hepatocellular carcinoma.

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