Supplementary Figure 2 from Premature Cell Cycle Entry Induced by Hepatitis B Virus Regulatory HBx Protein during Compensatory Liver Regeneration

Supplementary Figure 1 from Premature Cell Cycle Entry Induced by Hepatitis B Virus Regulatory HBx Protein during Compensatory Liver Regeneration

Background & Aims: Development of new and more effective therapies against hepatitis B virus (HBV) is limited by the lack of suitable small animal models. The HBV transgenic mouse model containing an integrated overlength 1.3-mer construct has yielded crucial insights, but this model unfortunately lacks covalently closed circular DNA (cccDNA), the episomal HBV transcriptional template, and cannot be cured given that HBV is integrated in every cell. Methods: To solve these 2 problems, we generated a novel transgenic mouse (HBV1.1X), which generates an excisable circular HBV genome using Cre/LoxP technology. This model possesses a HBV1.1-mer cassette knocked into the ROSA26 locus and is designed for stable expression of viral proteins from birth, like the current HBV transgenic mouse model, before genomic excision with the introduction of Cre recombinase. Results: We demonstrated induction of recombinant cccDNA (rcccDNA) formation via viral or transgenic Cre expression in HBV1.1X mice, and the ability to regulate HBsAg and HBc expression with Cre in mice. Tamoxifen-inducible Cre could markedly downregulate baseline HBsAg levels from the integrated HBV genome. To demonstrate clearance of HBV from HBV1.1X mice, we administered adenovirus expressing Cre, which permanently and significantly reduced HBsAg and core antigen levels in the murine liver via rcccDNA excision and a subsequent immune response. Conclusions: The HBV1.1X model is the first Cre-regulatable HBV transgenic mouse model and should be of value to mimic chronic HBV infection, with neonatal expression and tolerance of HBV antigens, and on-demand modulation of HBV expression. Lay summary: Hepatitis B virus (HBV) can only naturally infect humans and chimpanzees. Mouse models have been developed with the HBV genome integrated into mouse chromosomes, but this prevents mice from being cured. We developed a new transgenic mouse model that allows for HBV to be excised from mouse chromosomes to form a recombinant circular DNA molecule resembling the natural circular HBV genome. HBV expression could be reduced in these mice, enabling curative therapies to be tested in this new mouse model.

The hepatitis B virus (HBV) regulatory HBx protein is required for infection, and its binding to cellular damaged DNA binding protein 1 (DDB1) is critical for this function. DDB1 is an adaptor protein for the cullin 4A Really Interesting New Gene (RING) E3 ubiquitin ligase (CRL4) complex and functions by binding cellular DDB1 cullin associated factor (DCAF) receptor proteins that recruit substrates for ubiquitination and degradation. We compared the proteins found in the CRL4 complex immunoprecipitated from uninfected versus HBV-infected hepatocytes from human liver chimeric mice for insight into mechanisms by which HBV and the cell interact within the CRL4 complex. Consistent with its role as a viral DCAF, HBx was found in the HBV CRL4 complexes. In tissue culture transfection experiments, we showed that HBx expression led to decreased levels of known restriction factor structural maintenance of chromosomes protein 6 (SMC6) and putative restriction factors stromal interaction molecule 1 (STIM1, zinc finger E-box binding homeobox 2 (ZEB2), and proteasome activator subunit 4 (PSME4). Moreover, silencing of these proteins led to increased HBV replication in the HepG2-sodium taurocholate cotransporting polypeptide (NTCP) infection model. We also identified cellular DCAF receptors in CRL4 complexes from humanized mice. Increasing amounts of HBx did not reveal competitive DCAF binding to cullin4 (CUL4)-DDB1 in plasmid-transfected cells. Our results suggest a model in which HBx benefits virus replication by directly or indirectly degrading multiple cellular restriction factors.

BRIT1 has emerged as a novel key player in homologous recombination (HR). It is located in 8p23, a locus frequently deleted in hepatocellular carcinoma (HCC). Previously, we found that BRIT1-deficiency triggered genomic instability and tumor formation in our mouse model. Here we aim to determine whether BRIT1 aberrations are implicated in HCC and, if so, whether they can be used for targeted therapy with PARP inhibitors and other agents. We analyzed HCC samples for BRIT1 alterations at DNA, RNA and protein levels. BRIT1 was found deleted and/or downregulated in ~30% of HCC samples; BRIT1 mutant K659fsX10 identified in HCC abolished DNA repair function. Notably, BRIT1 deletion was correlated with poor survival and high recurrence of HCC. To determine the role of BRIT1 deficiency in potentiating the drug response, we subsequently generated BRIT1-deficient HCC cells, determined their HR defects, and assessed their response to the PARPi olaparib and PI3K inhibitor in vitro and in mice. BRIT1-deficient HCC cells were HR defective and hypersensitive to olaparib alone or in combination with PI3K inhibitor BEZ235, both in vitro and in vivo. The cytotoxicity of olaparib alone or in combination with BEZ235 was largely alleviated by ectopic BRIT1. We also found that BEZ235 markedly enhanced the production of poly (ADP-ribose) and the level of double-strand breaks (DSB) and single-strand breaks (SSB) in BRIT1-deficient cells. In summary, our results identify BRIT1 deficiency as a potential driver for HCC development, and BRIT1 status is critical to sensitivity to treatment with olaparib and/or BEZ235. PI3K inhibition induces substantial DNA damage and makes cells more dependent on PARP activity in the context of BRIT1 deficiency, thus, BRIT1 depletion facilitates enhancing synthetic lethality of PARP inhibitors and PI3K inhibitors in HCC. This study provides a new mechanistic foundation for significantly expanding the application of PARPi in HCC therapy.

ABSTRACT Five matching sets of nonmalignant liver tissues and hepatocellular carcinoma (HCC) samples from individuals chronically infected with hepatitis B virus (HBV) were examined. The HBV genomic sequences were determined by using overlapping PCR amplicons covering the entire viral genome. Four pairs of tissues were infected with HBV genotype C, while one pair was infected with HBV genotype B. HBV replication markers were found in all tissues. In the majority of HCC samples, the levels of pregenomic/precore RNA (pgRNA) and covalently closed circular DNA (cccDNA) were lower than those in liver tissue counterparts. Regardless of the presence of HBV replication markers, (i) integrant-derived HBV RNAs (id-RNAs) were found in all tissues by reverse transcription-PCR (RT-PCR) analysis and were considerably abundant or predominant in 6/10 tissue samples (2 liver and 4 HCC samples), (ii) RNAs that were polyadenylated using the cryptic HBV polyadenylation signal and therefore could be produced by HBV replication or derived from integrated HBV DNA were found in 5/10 samples (3 liver and 2 HCC samples) and were considerably abundant species in 3/10 tissues (2 livers and 1 HCC), and (iii) cccDNA-transcribed RNAs polyadenylated near position 1931 were not abundant in 7/10 tissues (2 liver and 5 HCC samples) and were predominant in only two liver samples. Subsequent RNA sequencing analysis of selected liver/HCC samples also showed relative abundance of id-RNAs in most of the examined tissues. Our findings suggesting that id-RNAs could represent a significant source of HBV envelope proteins, which is independent of viral replication, are discussed in the context of the possible contribution of id-RNAs to the HBV life cycle. IMPORTANCE The relative abundance of integrant-derived HBV RNAs (id-RNAs) in chronically infected tissues suggest that id-RNAs coding for the envelope proteins may facilitate the production of a considerable fraction of surface antigens (HBsAg) in infected cells bearing HBV integrants. If the same cells support HBV replication, then a significant fraction of assembled HBV virions could bear id-RNA-derived HBsAg as a major component of their envelopes. Therefore, the infectivity of these HBV virions and their ability to facilitate virus cell-to-cell spread could be determined mainly by the properties of id-RNA-derived envelope proteins and not by the properties of replication-derived HBsAg. These interpretations suggest that id-RNAs may play a role in the maintenance of chronic HBV infection and therefore contribute to the HBV life cycle. Furthermore, the production of HBsAg from id-RNAs independently of viral replication may explain at least in part why treatment with interferon or nucleos(t)ides in most cases fails to achieve a loss of serum HBsAg.

Chronic hepatitis B virus infection is a significant risk factor for cirrhosis and hepatocellular carcinoma. The HBx protein is required for virus replication, but the lack of robust infection models has hindered our understanding of HBx functions that could be targeted for antiviral purposes. We briefly review three properties of HBx: its binding to DDB1 and its regulation of cell survival and metabolism, to illustrate how a single viral protein can have multiple effects in a cell. We propose that different functions of HBx are needed, depending on the changing hepatocyte environment encountered during a chronic virus infection, and that these functions might serve as novel therapeutic targets for inhibiting hepatitis B virus replication and the development of associated diseases.

Chronic hepatitis B virus infection is a major risk factor for hepatocellular carcinoma (HCC). Hepatitis B virus X protein (HBx) is a hepatitis B virus protein that has multiple cellular functions, but its role in HCC pathogenesis has been controversial. Farnesoid X receptor (FXR) is a nuclear receptor with activities in anti‐inflammation and inhibition of hepatocarcinogenesis. However, whether or how FXR can impact hepatitis B virus/HBx‐induced hepatocarcinogenesis remains unclear. In this study, we showed that HBx can interact with FXR and function as a coactivator of FXR. Expression of HBx in vivo enhanced FXR‐responsive gene regulation. HBx also increased the transcriptional activity of FXR in a luciferase reporter gene assay. The HBx–FXR interaction was confirmed by coimmunoprecipitation and glutathione S‐transferase pull‐down assays, and the FXR activation function 1 domain was mapped to bind to the third α helix in the C terminus of HBx. We also found that the C‐terminally truncated variants of HBx, which were found in clinical HCC, were not effective at transactivating FXR. Interestingly, recruitment of the full‐length HBx, but not the C‐terminally truncated HBx, enhanced the binding of FXR to its response element. In vivo, FXR ablation markedly sensitized mice to HBx‐induced hepatocarcinogenesis. Conclusions: We propose that transactivation of FXR by full‐length HBx may represent a protective mechanism to inhibit HCC and that this inhibition may be compromised upon the appearance of C‐terminally truncated HBx or when the expression and/or activity of FXR is decreased. (Hepatology 2017;65:893‐906)

Background and aims: Understanding the molecular pathogenesis of hepatocellular carcinoma (HCC) is essential to identify therapeutic targets. A hepatitis B virus (HBV) related double transgenic murine model was developed.Methods: Liver specific expression of HBV X protein (HBx) and insulin receptor substrate 1 (IRS1) was achieved and transgenic mice were followed from birth to age 21 months. Liver and tumor tissue were assessed for histologic changes as well as activation of signal transduction pathways by qRT-PCR and multiplex ELM protein assays.Results: Overexpression of HBx and IRS1 stimulates liver cell proliferation in the double transgenic mice. Only the male mice developed HCC starting at age 15-18 months. The IN/IGF1/IRS1/MAPK/ERK and IN/IGF1/IRS1/PI3K/AKT/GSK3 beta cascades were activated early (6-9 months) in the liver followed by WNT/beta-catenin and Notch signaling. Aspartate beta-hydroxylase (ASPH) was found to link these upstream growth factor signaling pathways to downstream Notch activation in tumor tissues.Conclusions: Sustained overexpression of HBx and IRS1 led to constitutive activation of a tripartite growth factor signal transduction cascade in the liver and was necessary and sufficient to promote HCC development and progression. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

The efficient replication of hepatitis B virus (HBV) requires the HBV regulatory hepatitis B virus X (HBx) protein. The exact contributions of HBx are not fully understood, in part because of the limitations of the assays used for its study. When HBV replication is driven from a plasmid DNA, the contribution of HBx is modest. However, there is an absolute requirement for HBx in assays that recapitulate the infectious virus life cycle. There is much evidence that HBx can contribute directly to HBV replication by acting on viral promoters embedded within protein coding sequences. In addition, HBx may also contribute indirectly by modulating cellular pathways to benefit virus replication. Understanding the mechanism(s) of HBx action during virus replication may provide insight into novel ways to disrupt chronic HBV replication.

Chronic infection with hepatitis B virus (HBV) is a risk factor for developing hepatocellular carcinoma (HCC). The life cycle of HBV is complex and has been difficult to study because HBV does not infect cultured cells. The HBV regulatory X protein (HBx) controls the level of HBV replication and possesses an HCC cofactor role. Attempts to understand the mechanism(s) that underlie HBx effects on HBV replication and HBV‐associated carcinogenesis have led to many reported HBx activities that are likely influenced by the assays used. This review summarizes experimental systems commonly used to study HBx functions, describes limitations of these experimental systems that should be considered, and suggests approaches for ensuring the biological relevance of HBx studies. (Hepatology 2015;61:1416–1424)

Diverse epidemiological factors are associated with hepatocellular carcinoma (HCC) prevalence in different populations. However, the global landscape of the genetic changes in HCC genomes underpinning different epidemiological and ancestral backgrounds still remains uncharted. Here a collection of data from 503 liver cancer genomes from different populations uncovered 30 candidate driver genes and 11 core pathway modules. Furthermore, a collaboration of two large-scale cancer genome projects comparatively analyzed the trans-ancestry substitution signatures in 608 liver cancer cases and identified unique mutational signatures that predominantly contribute to Asian cases. This work elucidates previously unexplored ancestry-associated mutational processes in HCC development. A combination of hotspot TERT promoter mutation, TERT focal amplification and viral genome integration occurs in more than 68% of cases, implicating TERT as a central and ancestry-independent node of hepatocarcinogenesis. Newly identified alterations in genes encoding metabolic enzymes, chromatin remodelers and a high proportion of mTOR pathway activations offer potential therapeutic and diagnostic opportunities.

The hepatitis B virus (HBV) causes acute and chronic hepatitis, and the latter is a major risk factor for the development of hepatocellular carcinoma (HCC). HBV encodes a 17-kDa regulatory protein, HBx, which is required for virus replication. Although the precise contribution(s) of HBx to virus replication is unknown, many viruses target cellular pathways to create an environment favorable for virus replication. The ubiquitin proteasome system (UPS) is a major conserved cellular pathway that controls several critical processes in the cell by regulating the levels of proteins involved in cell cycle, DNA repair, innate immunity, and other processes. We summarize here the interactions of HBx with components of the UPS, including the CUL4 adaptor DDB1, the cullin regulatory complex CSN, and the 26S proteasome. Understanding how these protein interactions benefit virus replication remains a challenge due to limited models in which to study HBV replication. However, studies from other viral systems that similarly target the UPS provide insight into possible strategies used by HBV.

Abstract Hepatocellular carcinoma (HCC) is one of the most prevalent human cancers, especially in Asia, and regions of endemic hepatitis infection. The primary risk factors for HCC are chronic hepatitis infection by hepatitis C (HCV) or hepatitis B (HBV). We performed whole exome sequencing on 549 matched tumor and normal samples from three centers (BCM HGSC, RCAST, NCC) to identify somatic changes in protein coding regions and changes in copy number. Here we describe variants associated with specific viral etiologies in HCC. We found TP53, CTNNB1, ARID1A, ARID2, and AXIN1 to be among the most frequently mutated genes in the entire dataset, implicating DNA damage, WNT signaling, and chromatin remodeling as recurrently altered pathways in HCC. Our validation rate for mutations in significantly mutated genes was over 95%. We identified 18 genes which differed significantly (p < 0.05) in mutation frequency between viral subtypes. Of these, AXIN1 was mutated in over 15% of HBV-associated HCC but less than 5% of HCV and non-viral-associated HCC. Additionally, we performed GISTIC analysis based on read depth from our whole exome sequencing data. We found that WNT3A and MYC were significantly amplified and AXIN1 and TP53 were significantly deleted in HCV-associated HCC but not HBV or non-virus-associated HCC. Our findings indicate that WNT/beta-catenin signaling is a frequently altered pathway in HCC. While aberrations in this pathway were present in all viral etiologies, HBV-associated HCC tended to mutate AXIN1 while HCV-associated HCC tended to have AXIN1 deletion. These differences may result from virus-specific oncogenic processes. Citation Format: Kyle R. Covington, Lawrence A. Donehower, Chad Creighton, Betty L. Slagle, John A. Goss, Ronald T. Cotton, Marie-Claude Gingras, Eve Shinbrot, Jacfranz J. Guiteau, Thao N. Nguyen, Theresa R. Harring, Donna M. Muzny, Kimberly Walker, HarshaVardhan Doddapaneni, Richard A. Gibbs, Hiroyuki Aburatani, Tatsuhiro Shibata, David A. Wheeler, Japan ICGC HCC Project. Viral subtypes in hepatocellular carcinoma are associated with different mechanisms of WNT/CTNNB1 alteration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2211. doi:10.1158/1538-7445.AM2014-2211

Robust hepatitis B virus (HBV) replication is stimulated by the regulatory HBx protein. HBx binds the cellular protein DDB1; however, the importance of this interaction for HBV replication remains unknown. We tested whether HBx binding to DDB1 was required for HBV replication using a plasmid based replication assay in HepG2 cells. Three DDB1 binding-deficient HBx point mutants (HBx(69), HBx(90/91), HBx(R96E)) failed to restore wildtype levels of replication from an HBx-deficient plasmid, which established the importance of the HBx-DDB1 interaction for maximal HBV replication. Analysis of overlapping HBx truncation mutants revealed that both the HBx-DDB1 binding domain and the carboxyl region are required for maximal HBV replication both in vitro and in vivo, suggesting the HBx-DDB1 interaction recruits regulatory functions critical for replication. Finally we demonstrate that HBx localizes to the Cul4A-DDB1 complex, and discuss the possible implications for models of HBV replication.

Hepatitis B virus (HBV) infection is an important risk factor for hepatocellular carcinoma (HCC). The hepatitis B virus X protein (HBx), a multifunctional regulatory protein encoded by HBV, is known to be involved in stimulation of viral replication by modulating cell cycle status. HBx is required for maximal virus replication in plasmid-based replication assays in immortalized human liver HepG2 cells and in primary rat hepatocytes. Moreover, the C-terminal region of HBx is important for HBV replication in HepG2 cells. However, in normal hepatocytes, the region of HBx that is responsible for its effect on cell cycle regulation and HBV replication is unclear. We have demonstrated that HBx is similarly required for maximal HBV replication in primary mouse hepatocytes and that the C-terminus of HBx is essential for its ability to stimulate HBV replication by inducing quiescent hepatocytes to exit G0 phase of the cell cycle but stall in G1 phase. Our studies establish that primary mouse hepatocytes support HBx-dependent HBV replication, and provide further evidence for the effect of the C-terminal region of HBx on HBV infection and replication.

ABSTRACT Hepatitis B virus (HBV) encodes the regulatory HBx protein, which is required for virus replication, although its specific role(s) in the replication cycle remains under investigation. An immunoprecipitation/mass spectrometry approach was used to identify four novel HBx binding proteins from the cytoplasmic fraction of HBx transgenic mouse livers. One of these HBx binding partners is beta interferon promoter stimulator 1 (IPS-1), an adaptor protein that plays a critical role in mediating retinoic acid-inducible gene I (RIG-I) signaling, which leads to the activation of beta interferon (IFN-β). The HBx-IPS-1 protein interaction was confirmed in plasmid-transfected HepG2 cells by reciprocal coimmunoprecipitation and Western blotting. We hypothesized that HBx might alter IPS-1 function since proteins of hepatitis C virus and hepatitis A virus similarly bind IPS-1 and target it for inactivation. The effect of HBx on IPS-1-mediated IFN-β signaling was tested in transfected 293T and HepG2 cells, and we show that HBx inhibits double-stranded DNA (dsDNA)-mediated IFN-β activation in a dose-dependent manner when expressed either alone or within the context of HBV replication. However, HBx does not inhibit poly(I:C)-activated IFN-β signaling. These results demonstrate that HBx interferes with the RIG-I pathway of innate immunity. Hepatitis B virus now joins hepatitis C virus and hepatitis A virus in targeting the same innate immune response pathway, presumably as a shared strategy to benefit replication of these viruses in the liver.

Identifying the requirements for the regulatory HBx protein in hepatitis B virus (HBV) replication is an important goal. A plasmid-based HBV replication assay was used to evaluate whether HBx subcellular localization influences its ability to promote virus replication, as measured by real time PCR quantitation of viral capsid-associated DNA. HBx targeted to the nucleus by a nuclear localization signal (NLS-HBx) was able to restore HBx-deficient HBV replication, while HBx containing a nuclear export signal (NES-HBx) was not. Both NLS-HBx and NES-HBx were expressed at similar levels (by immunoprecipitation and Western blotting), and proper localization of the signal sequence-tagged proteins was confirmed by deconvolution microscopy using HBx, NLS-HBx, and NES-HBx proteins fused to GFP. Importantly, these findings were confirmed in vivo by hydrodynamic injection into mice. Our results demonstrate that in these HBV replication assays, at least one function of HBx requires its localization to the nucleus.