Hepatitis B Virus X protein is essential to initiate and maintain virus replication after infection

Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Trogerstrasse, 30, 81675 Munich, Germany.
Journal of Hepatology (Impact Factor: 11.34). 03/2011; 55(5):996-1003. DOI: 10.1016/j.jhep.2011.02.015
Source: PubMed


The molecular biology of hepatitis B virus (HBV) has been extensively studied but the exact role of the hepatitis B X protein (HBx) in the context of natural HBV infections remains unknown.
Primary human hepatocytes and differentiated HepaRG cells allowing conditional trans complementation of HBx were infected with wild type (HBV(wt)) or HBx deficient (HBV(x-)) HBV particles and establishment of HBV replication was followed.
We observed that cells inoculated with HBx-deficient HBV particles (HBV(x-)) did not lead to productive HBV infection contrary to cells inoculated with wild type HBV particles (HBV(wt)). Although equal amounts of nuclear covalently closed circular HBV-DNA (cccDNA) demonstrated comparable uptake and nuclear import, active transcription was only observed from HBV(wt) genomes. Trans-complementation of HBx was able to rescue transcription from the HBV(x-) genome and led to antigen and virion secretion, even weeks after infection. Constant expression of HBx was necessary to maintain HBV antigen expression and replication. Finally, we demonstrated that HBx is not packaged into virions during assembly but is expressed after infection within the new host cell to allow epigenetic control of HBV transcription from cccDNA.
Our results demonstrate that HBx is required to initiate and maintain HBV replication and highlight HBx as the key regulator during the natural infection process.

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    • "HBV inoculum was either concentrated from filtered HepG2.2.15 (wild-type virus) or K6 (HBx negative virus) [25] supernatants by polyethylene glycol (PEG) precipitation as previously described [22], or partially purified by heparin chromatography [26], then concentrated using centrifugal filters devices (Amicon Ultracel 100K, Millipore). A mock ''HBV-negative'' inoculum (mock control) was generated by depletion of Dane particles, HBsAg and HBeAg using centrifugal filters devices (Amicon Ultracel 10K, Millipore). "
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    ABSTRACT: The outcome of Hepatitis B virus infection may be influenced by early interactions between the virus and hepatocyte innate immune responses. To date, the study of such interactions during the very early step of infection has not been adequately investigated. We used the HepaRG cell line, as well as primary human hepatocytes to analyze, within 24 hours of exposure to HBV, either delivered by a physiologic route or baculovirus vector (Bac-HBV), the early modulation of the expression of selected antiviral/pro-inflammatory cytokines and interferon-stimulated-genes. Experiments were also performed in presence or absence of innate receptor agonists to investigate early HBV-induced blockade of innate responses. We show that hepatocytes themselves could detect HBV, and express innate genes when exposed to either HBV virions or Bac-HBV. Whereas Bac-HBV triggered a strong antiviral cytokine secretion followed by the clearance of replicative intermediates, a physiologic HBV exposure led to an abortive response. The early inhibition of innate response by HBV was mainly evidenced on TLR3 and RIG-I/MDA5 signaling pathways upon engagement with exogenous agonist, leading to a decreased expression of several pro-inflammatory and antiviral cytokine genes. Finally, we demonstrate that this early inhibition of dsRNA-mediated response is due to factor(s) present in the HBV inoculum, but not being HBsAg or HBeAg themselves, and does not require de novo viral protein synthesis and replication. Our data provide strong evidence that HBV viral particles themselves can readily inhibit host innate immune responses upon virion/cell interactions, and may explain, at least partially, the "stealthy" character of HBV. Copyright © 2015. Published by Elsevier B.V.
    Journal of Hepatology 07/2015; DOI:10.1016/j.jhep.2015.07.014 · 11.34 Impact Factor
    • "Thus, cccDNA is persistent throughout the cycle of infectivity. cccDNA is decorated with the usual proteins of euchromatin with the addition of two HBV proteins: X and Cp (Belloni et al., 2009; Bock et al., 2001; Buendia and Neuveut, 2015; Ducroux et al., 2014; Fallot et al., 2012; Levrero et al., 2009; Lucifora et al., 2011; Pollicino et al., 2006). "
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    ABSTRACT: Hepatitis B Virus (HBV) is a small virus whose genome has only four open reading frames. We argue that the simplicity of the virion correlates with a complexity of functions for viral proteins. We focus on the HBV core protein (Cp), a small (183 residue) protein that self-assembles to form the viral capsid. However, its functions are a little more complicated than that. In an infected cell Cp modulates every step of the viral lifecycle. Cp is bound to nuclear viral DNA and affects its epigenetics. Cp correlates with RNA specificity. Cp assembles specifically on a reverse transcriptase-viral RNA complex or, apparently, nothing at all. Indeed Cp has been one of the model systems for investigation of virus self-assembly. Cp participates in regulation of reverse transcription. Cp signals completion of reverse transcription to support virus secretion. Cp carries both nuclear localization signals and HBV surface antigen (HBsAg) binding sites; both of these functions appear to be regulated by contents of the capsid. Cp can be targeted by antivirals - while self-assembly is the most accessible of Cp activities, we argue that it makes sense to engage the broader spectrum of Cp function. This article forms part of a symposium in Antiviral Research on "From the discovery of the Australia antigen to the development of new curative therapies for hepatitis B: an unfinished story." Copyright © 2015. Published by Elsevier B.V.
    Antiviral research 06/2015; 121. DOI:10.1016/j.antiviral.2015.06.020 · 3.94 Impact Factor
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    • "Recent study also showed that the truncation of X protein may result in the inhibition of Core protein although the level of cccDNA didn't change by sgRNA targeting HBx protein (Seeger and Sohn, 2014). It is possible because HBx takes an important role in transcription of preC/C RNA from episomal cccDNA templates (Belloni et al., 2009; Lucifora et al., 2011). Considering the highly economical use of HBV genetic material, whether the inhibition effects caused by frame shift of direct mutagenesis or truncated HBx transcriptional inaction need to be further investigated. "
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    ABSTRACT: Chronic hepatitis B virus (HBV) infection causes livercirrhosis and hepatocellular carcinoma and remains a serious health problem worldwide. Covalently closed circular DNA (cccDNA) in the liver cell nucleus sustains HBV infection. Major treatments for HBV infection include the use of interferon-α and nucleotide analogs, but they cannot eradicate cccDNA. As a novel tool for genome editing, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system developed from bacteria can be used to accurately and efficiently engineer and modify genomic DNA. In this study, the CRISPR/Cas9 system was used to target the HBV genome and efficiently inhibit HBV infection. We synthesized four single-guide RNAs (sgRNAs) targeting the conserved regions of HBV. The expression of these sgRNAS with Cas9 reduced the viral production in Huh7 cells as well as in HBV-replication cell HepG2.2.15. We further demonstrated that CRISPR/Cas9 direct cleavage and cleavage-mediated mutagenesis occurred in HBV cccDNA of transfected cells. In the new mouse model carrying HBV cccDNA, injection of sgRNA-Cas9 plasmids viarapidtailveinresulted in the low level of cccDNA and HBV protein. In conclusion, the designed CRISPR/Cas9 system can accurately and efficiently target HBV cccDNA and inhibit HBV replication. This system may be used as a novel therapeutic strategy against chronic HBV infection. Copyright © 2015. Published by Elsevier B.V.
    Antiviral research 04/2015; 118. DOI:10.1016/j.antiviral.2015.03.015 · 3.94 Impact Factor
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