Hepatitis B Virus Replication System Research Articles

Multiomics interrogation into HBV (Hepatitis B virus)-host interaction reveals novel coding potential in human genome, and identifies canonical and non-canonical proteins as host restriction factors against HBV

Hepatitis B Virus (HBV) constitutes a major threat to global public health. Current understanding of HBV-host interaction is yet limited. Here, ribosome profiling, quantitative mass spectrometry and RNA-sequencing were conducted on a recently established HBV replication system, through which we identified multiomic differentially expressed genes (DEGs) that HBV orchestrated to remodel host proteostasis networks. Our multiomics interrogation revealed that HBV induced significant changes in both transcription and translation of 35 canonical genes including PPP1R15A, PGAM5 and SIRT6, as well as the expression of at least 15 non-canonical open reading frames (ncORFs) including ncPON2 and ncGRWD1, thus revealing an extra coding potential of human genome. Overexpression of these five genes but not the enzymatically deficient SIRT6 mutants suppressed HBV replication while knockdown of SIRT6 had opposite effect. Furthermore, the expression of SIRT6 was down-regulated in patients, cells or animal models of HBV infection. Mechanistic study further indicated that SIRT6 directly binds to mini-chromosome and deacetylates histone H3 lysine 9 (H3K9ac) and histone H3 lysine 56 (H3K56ac), and chemical activation of endogenous SIRT6 with MDL800 suppressed HBV infection in vitro and in vivo. By generating the first multiomics landscape of host-HBV interaction, our work is thus opening a new avenue to facilitate therapeutic development against HBV infection.

Proteasomal activator 28 gamma stabilizes hepatitis B virus X protein by competitively inhibiting the Siah-1-mediated proteasomal degradation

Proteasomal activator 28 gamma (PA28γ) upregulates the levels of HBx, a regulatory protein of hepatitis B virus (HBV) to stimulate HBV replication; however, the detailed mechanism remains unknown. Here, we found that PA28γ impaired the ability of seven in absentia homolog 1 (Siah-1) as an E3 ubiquitin ligase of HBx. PA28γ competitively inhibited the binding of Siah-1 to HBx in human hepatoma cells. Accordingly, PA28γ increased the stability of HBx and decreased HBx ubiquitination, abolishing the potential of Siah-1 to downregulate HBx levels. PA28γ also executed its role as an antagonist of Siah-1 during HBV replication, as demonstrated by an in vitro HBV replication system. The present study may provide insights into the mechanisms underlying the regulation of HBV replication.

Repurposing of Antazoline Hydrochloride as an Inhibitor of Hepatitis B Virus DNA Secretion.

Hepatitis B virus (HBV) belongs to Hepadnaviridae family and mainly infects hepatocytes, which can cause acute or chronic hepatitis. Currently, two types of antiviral drugs are approved for chronic infection clinically: interferons and nucleos(t)ide analogues. However, the clinical cure for chronic infection is still rare,and it is a huge challenge for all researchers to develop high-efficiency, safe, non-tolerant, and low-toxicity anti-HBV drugs. Antazoline hydrochloride is a first-generation antihistamine with anticholinergic properties, and it is commonly used to relieve nasal congestion and in eye drops. Recently, an in vitro high-throughput evaluation system was constructed to screen nearly 800 compounds from the Food and Drug Administration (FDA)-approved Drug Library. We found that arbidol hydrochloride and antazoline hydrochloride can effectively reduce HBV DNA in the extracellular supernatant in a dose-dependent manner, with EC50 of 4.321μmol/L and 2.910μmol/L in HepAD38 cells, respectively. Moreover, the antiviral effects and potential mechanism of action of antazoline hydrochloride were studied in different HBV replication systems. The results indicate that antazoline hydrochloride also has a significant inhibitory effect on HBV DNA in the extracellular supernatant of Huh7 cells, with an EC50 of 2.349μmol/L. These findings provide new ideas for screening and research related to HBV agents.

Establishment of a fluorescent in situ hybridization assay for imaging hepatitis B virus nucleic acids in cell culture models

While chronic hepatitis B remains a global public health problem, the detailed spatiotemporal dynamics of the key molecular events leading to the multiplication and egress of hepatitis B virus (HBV) are still largely unclear. Previously, we developed a chromogenic in situ hybridization assay for detection of HBV RNA, DNA and covalently closed circular DNA in clinical liver biopsies. Here, we report the establishment of a fluorescent in situ hybridization method for the visualization of HBV RNA, HBV core particle DNA and intranuclear DNA in a tetracycline-inducible HBV replication system (HepAD38) and a de novo infection system (HepG2-NTCP). Using 3D-STORM (three-dimensional stochastic optical reconstruction microscopy), we were able to obtain images of HBV RNA and DNA with improved spatial resolution allowing in-depth analyses of key virological events within complex subcellular compartments. Taken together, these techniques should facilitate a deeper understanding of the molecular events of the HBV life cycle and shed new light on the intricate mechanisms of virus–host interactions.Emerging Microbes & Infections (2017) 6, e98; doi:10.1038/emi.2017.84; published online 8 November 2017

DDX3 DEAD-box RNA helicase is a host factor that restricts hepatitis B virus replication at the transcriptional level.

DDX3 is a member of the DEAD-box RNA helicase family, involved in mRNA metabolism, including transcription, splicing, and translation. We previously identified DDX3 as a hepatitis B virus (HBV) polymerase (Pol) binding protein, and by using a transient transfection, we found that DDX3 inhibits HBV replication at the posttranscriptional level, perhaps following encapsidation. To determine the exact mechanism of the inhibition, we here employed a diverse HBV experimental system. Inconsistently, we found that DDX3-mediated inhibition occurs at the level of transcription. By using tetracycline-inducible HBV-producing cells, we observed that lentivirus-mediated DDX3 expression led to a reduced level of HBV RNAs. Importantly, knockdown of DDX3 by short hairpin RNA resulted in augmentation of HBV RNAs in two distinct HBV replication systems: (i) tetracycline-inducible HBV-producing cells and (ii) constitutive HBV-producing HepG2.2.15 cells. Moreover, DDX3 knockdown in HBV-susceptible HepG2-NTCP cells, where covalently closed circular DNA (cccDNA) serves as the template for viral transcription, resulted in increased HBV RNAs, validating that transcription regulation by DDX3 occurs on a physiological template. Overall, our results demonstrate that DDX3 represents an intrinsic host antiviral factor that restricts HBV transcription. Upon entry into host cells, viruses encounter host factors that restrict viral infection. During evolution, viruses have acquired the ability to subvert cellular factors that adversely affect their replication. Such host factors include TRIM5α and APOBEC3G, which were discovered in retroviruses. The discovery of host restriction factors provided deeper insight into the innate immune response and viral pathogenesis, leading to better understanding of host-virus interactions. In contrast to the case with retroviruses, little is known about host factors that restrict hepatitis B virus (HBV), a virus distantly related to retroviruses. DDX3 DEAD box RNA helicase is best characterized as an RNA helicase involved in RNA metabolism, such as RNA processing and translation. Here, we show that DDX3 inhibits HBV infection at the level of viral transcription.

Nuclear Export and Import of Human Hepatitis B Virus Capsid Protein and Particles

It remains unclear what determines the subcellular localization of hepatitis B virus (HBV) core protein (HBc) and particles. To address this fundamental issue, we have identified four distinct HBc localization signals in the arginine rich domain (ARD) of HBc, using immunofluorescence confocal microscopy and fractionation/Western blot analysis. ARD consists of four tight clustering arginine-rich subdomains. ARD-I and ARD-III are associated with two co-dependent nuclear localization signals (NLS), while ARD-II and ARD-IV behave like two independent nuclear export signals (NES). This conclusion is based on five independent lines of experimental evidence: i) Using an HBV replication system in hepatoma cells, we demonstrated in a double-blind manner that only the HBc of mutant ARD-II+IV, among a total of 15 ARD mutants, can predominantly localize to the nucleus. ii) These results were confirmed using a chimera reporter system by placing mutant or wild type HBc trafficking signals in the heterologous context of SV40 large T antigen (LT). iii) By a heterokaryon or homokaryon analysis, the fusion protein of SV40 LT-HBc ARD appeared to transport from nuclei of transfected donor cells to nuclei of recipient cells, suggesting the existence of an NES in HBc ARD. This putative NES is leptomycin B resistant. iv) We demonstrated by co-immunoprecipitation that HBc ARD can physically interact with a cellular factor TAP/NXF1 (Tip-associated protein/nuclear export factor-1), which is known to be important for nuclear export of mRNA and proteins. Treatment with a TAP-specific siRNA strikingly shifted cytoplasmic HBc to nucleus, and led to a near 7-fold reduction of viral replication, and a near 10-fold reduction in HBsAg secretion. v) HBc of mutant ARD-II+IV was accumulated predominantly in the nucleus in a mouse model by hydrodynamic delivery. In addition to the revised map of NLS, our results suggest that HBc could shuttle rapidly between nucleus and cytoplasm via a novel TAP-dependent NES.

Proteomic analysis of HBV-associated HCC: Insights on mechanisms of disease onset and biomarker discovery

The development of hepatocellular carcinoma (HCC) can be considered as an end-stage outcome of chronic hepatitis B virus (HBV) infection. Early prognostic markers are needed to allow effective treatments and prevent HCC from developing. Proteomics analysis has been used to identify markers from clinical samples from HCC patients. This approach can be further improved by identifying early biomarkers before the onset of HCC. One way would be to use the cell-based HBV replication system, which is reflective of the early stage of virus infection and thus secreted proteins identified at this stage may have relevance in HCC prognosis. In this review, we focus the discussion on the current status of proteomics analysis of cellular proteins and HCC biomarker identification, with a special highlight on the potential of the cell-based HBV replication system for the identification of prognostic HCC biomarkers.

ITRAQ-Coupled 2D LC–MS/MS Analysis of Secreted Proteome of HBV-Replicating HepG2 Cells: Potential in Biomarkers for Prognosis of HCC

Hepatitis B virus (HBV) infection remains a major health concern with more than 350 million carriers in the world. It is associated with acute and chronic liver diseases including hepatocellular carcinoma (HCC). The early detection of severe liver diseases related to HBV is crucial for the effective treatment. This work aims to investigate the secreted proteins in our recently established cell-based HBV replication system, using isobaric tags for relative and absolute quantitation (iTRAQ)-coupled 2D LC-MS/MS proteomics approach. Such proteins are reflective of early events of HBV infection and thus may have potential as prognostic biomarkers for development of liver diseases.

Rac1 GTPase is activated by hepatitis B virus replication — involvement of HBX

Hepatitis B virus (HBV) is a causative agent for liver diseases including hepatocellular carcinoma. Understanding its interactions with cellular proteins is critical in the elucidation of the mechanisms of disease progression. Using a cell-based HBV replication system, we showed that HBV replication in HepG2 cells resulted in a cellular morphological changes displaying membrane rufflings and lamellipodia like structures reminiscent of cells expressing constitutively activated Rac1. We also showed that activated Rac1 resulted in increased viral replication. HBV replication specifically activated wild type Rac1, but not Cdc42. The Rac1 activation by HBV replication also resulted in the phosphorylation of ERK1/2 and AKT, the downstream targets of Rac1 signaling cascade. The smallest HBV viral protein, HBX, was able to activate the endogenous Rac1 and induce membrane ruffling when transfected into cells. Significantly, HBX was found to directly interact with a Rac1 nucleotide exchange factor (βPIX) through a SH3 binding motif. Taken together, we have shown the interaction of HBV with the Rho GTPase, affecting cell morphology through the Rac1 activation pathway. HBV may possibly make use of an activated Rac1 signaling pathway for increased replication and resultant metastatic effects.

O.020 HBV/HCV: Animal/cellular models

Hepatitis B virus (HBV) infection is highly specific to primates. To cross the species barrier, we injected naked plasmid DNA containing a 1.5-fold-overlength genome of HBV into immunocompetent mice via the tail vein with acute circulatory overload. The injection resulted in production of viral transcripts specifically in the liver and expression of hepatitis B core protein in approximately 3% of the hepatocytes. HBV was secreted into the blood, evidenced by the presence of DNase I-resistant HBV sequence in fractionated serum at a density of 1.21 g/ml. The HBV DNA positivity in the serum persisted for 1 week. Most mice became positive for hepatitis B surface antigen for 2 weeks and later seropositive for anti-hepatitis B surface antibody. This simple and efficient HBV replication system in mice could be useful for investigating whether viral mutations as well as host genetic background may affect viral replication and induction of immune response.

Interferon prevents formation of replication-competent hepatitis B virus RNA-containing nucleocapsids

We have previously shown that IFN-beta inhibits hepatitis B virus (HBV) replication by noncytolytic mechanisms that either destabilize pregenomic (pg)RNA-containing capsids or prevent their assembly. Using immortalized murine hepatocyte cell lines stably transfected with a doxycycline (dox)-inducible HBV replication system, we now show that replication-competent pgRNA-containing capsids are not produced when the cells are pretreated with IFN-beta before HBV expression is induced with dox. Furthermore, the turnover rate of preformed HBV RNA-containing capsids is not changed in the presence of IFN-beta or IFN-gamma under conditions in which further pgRNA synthesis is inhibited by dox removal. In summary, these results demonstrate that types 1 and 2 IFN activate hepatocellular mechanism(s) that prevent the formation of replication-competent HBV capsids and, thereby, inhibit HBV replication.

Activation and inhibition of cellular calcium and tyrosine kinase signaling pathways identify targets of the HBx protein involved in hepatitis B virus replication.

Human hepatitis B virus (HBV) HBx protein is a multifunctional protein that activates cellular signaling pathways and is thought to be essential for viral infection. Woodchuck HBV mutants that lack HBx are unable to replicate in vivo or are severely impaired. HBV replication in HepG2 cells, a human hepatoblastoma cell line, is stimulated 5- to 10-fold by HBx protein. We have utilized the HepG2, HBx-dependent HBV replication system to study the effects of activators and inhibitors of cytosolic calcium and tyrosine kinase signaling pathways on viral replication. By transfecting either a wild-type HBV genome or an HBV genome that does not express HBx and then treating transfected cells with activators or inhibitors of signaling pathways, we identified compounds that either impair wild-type HBV replication or rescue HBx-deficient HBV replication. Geldanamycin or herbimycin A, tyrosine kinase inhibitors, blocked HBV replication. Derivatives of cyclosporine, i.e., cyclosporine A, cyclosporine H, and SDZ NIM811, which block cytosolic calcium signaling and specifically the mitochondrial permeability transition pore (SDZ NIM811), also impaired HBV replication. Treatment of cells with compounds that increase cytosolic calcium levels by a variety of mechanisms rescued replication of an HBx-deficient HBV mutant. Transcription of viral RNA and production of viral capsids were only minimally affected by these treatments. These results define a functional signaling circuit for HBV replication that includes calcium signaling and activation of cytosolic signaling pathways involving Src kinases, and they suggest that these pathways are stimulated by HBx acting on the mitochondrial transition pore.

Intravenous injection of naked plasmid DNA encoding hepatitis B virus (HBV) produces HBV and induces humoral immune response in mice

Hepatitis B virus (HBV) infection is highly specific to primates. To cross the species barrier, we injected naked plasmid DNA containing a 1.5-fold-overlength genome of HBV into immunocompetent mice via the tail vein with acute circulatory overload. The injection resulted in production of viral transcripts specifically in the liver and expression of hepatitis B core protein in approximately 3% of the hepatocytes. HBV was secreted into the blood, evidenced by the presence of DNase I-resistant HBV sequence in fractionated serum at a density of 1.21 g/ml. The HBV DNA positivity in the serum persisted for 1 week. Most mice became positive for hepatitis B surface antigen for 2 weeks and later seropositive for anti-hepatitis B surface antibody. This simple and efficient HBV replication system in mice could be useful for investigating whether viral mutations as well as host genetic background may affect viral replication and induction of immune response.