Technical Standards for Hepatitis B Virus X protein (HBx) Research Betty L. Slagle1, Ourania M. Andrisani2, Michael J. Bouchard3, Caroline G. L. Lee4, J.-H. James Ou5, and Aleem Siddiqui6

1

Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, TX

77030; 2Department of Basic Medical Sciences and Purdue Center for Cancer Research, Purdue University, West Lafayette, IN 47907; 3Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102; 4Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119077, Singapore; Division of Medical Sciences, Humphrey Oei Institute of Cancer Research, National Cancer Centre Singapore, Singapore 169610, Singapore; Duke-NUS Graduate Medical School Singapore, Singapore 169547, Singapore; 5Department of Molecular Microbiology and Immunology, University of Southern California, Keck School of Medicine, Los Angeles, CA 90033; 6Division of Infectious Diseases, University of California, San Diego, CA 92093 Key Words: virus life cycle viral pathogenesis hepatocellular carcinoma hepatitis B virus hepatitis B virus regulatory X protein

This article has been accepted for publication and undergone full peer review but has not bee through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/hep.27360

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2 Footnotes Page Contact Information for corresponding author Betty L. Slagle, Ph.D. Department of Molecular Virology & Microbiology Baylor College of Medicine Mailstop BCM-385 Houston, TX 77030 USA Tel: 713-798-3006 Fax: 713-798-5075 Email: [email protected]

Abbreviations HBV, hepatitis B virus HBx, hepatitis B virus regulatory X protein HCC, hepatocellular carcinoma ORF, open reading frame cccDNA, covalently-closed circular DNA WHV, woodchuck hepatitis virus WHx, woodchuck hepatitis virus X protein

Financial Support: NIH CA177951 (BLS); NIH DK04453 (OA); AI085087, DK077704, and DK08379 (AS); CA177337 and DK100257 (JO).

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3 Abstract 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.

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4 The human hepatitis B virus (HBV) belongs to the Hepadnaviridae family of hepatotropic DNA viruses. HBV can cause acute or chronic infection of the liver, and the latter is a risk factor for severe liver diseases including hepatocellular carcinoma (HCC). Worldwide, 400 million people have a chronic HBV infection and are at risk for premature death due to liver disease (1). Current therapies for chronic HBV infection are inadequate, and there is need for discovery of new and effective antiviral therapies. The highly compact HBV genome contains 4 overlapping open reading frames (ORFs) that encode 7 proteins; viral transcriptional elements are embedded within ORFs (Fig. 1). Much attention has focused on the regulatory HBx protein, which plays a critical, but not fully understood, role in HBV replication and associated carcinogenesis. Lack of HBV infection models that use cultured cells and the unique properties of HBx have led to information about HBx function that is often controversial or of unproven physiological relevance. In this review, we summarize assays commonly used to study HBx function in HBV replication and HBVassociated tumorigenesis and suggest guidelines for experimental design. Our goal is to raise awareness of the advantages and disadvantages of specific experimental systems and to assist investigators studying HBx to generate a better understanding of the role(s) of HBx in HBV replication and HBV-mediated hepatocarcinogenesis. In this review, the term virus life cycle includes all events starting from infection of a susceptible cell to the release of progeny virus; virus replication refers to specific steps in the virus life cycle that can be studied outside the context of the virus life cycle. We apologize to colleagues whose papers are not cited in this brief review. HBx protein HBx is encoded by the smallest ORF of HBV and is essential for the virus life cycle. HBx also contributes to the development of HBV-associated HCC. Understanding the role(s) of HBx in the virus life cycle (Fig. 2) and in development of HBV-associated HCC remains a significant challenge. Available assay systems are technically challenging, and inherent differences in

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5 experimental systems have produced different HBx effects on the level of HBV replication. Use of different experimental systems and HBx expression levels has also led to differences in the effects of HBx on cellular signal transduction pathways that could influence HCC development. Consequently, the HBx literature describes HBx effects that are often conflicting, and the mechanism by which HBx functions in the HBV life cycle and during HBV-associated carcinogenesis is an intensely debated topic. For an overview of HBx functions in HBV replication and its proposed role(s) in HBV-associated HCC, the reader is referred to the following reviews (2, 3). Methods used to study HBx function in the HBV life cycle The inability to infect cultured cells with HBV, combined with its compact genome organization, prevents studies employing traditional genetic approaches. Various assays are currently used to study HBx (Table 1). By necessity, most in vitro studies of HBx functions during HBV replication have used HBx overexpression in cell culture systems, and HBx effects differ, depending on the cell type used and the HBx expression level attained. Below is a brief summary of these assays, with cautionary notes for potential pitfalls and when possible, ways to avoid these problems. Transient transfection with plasmids encoding HBx. Transfection of plasmid DNA encoding HBx into cultured cells has generated important information about HBx functions, including that it activates cellular signal transduction cascades, induces expression of cellular and viral genes, alters cell cycle progression, and sensitizes cells to apoptotic signals [reviewed in (2-4)]. HBx also interacts directly or indirectly with general transcription factors, DNA repair, and signal transduction molecules [reviewed in (2, 3, 5)]. HBx associates with mitochondria and induces fission and mitophagy (6), and binds chromatin modifiers to negatively regulate HBV transcription (7, 8). Limitations and recommendations. Expression of HBx by transfection provides information only for the function of HBx. To assure that identified HBx functions are biologically

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6 relevant, we recommend that results be confirmed in the context of HBV replication using assays described below. Such confirmation has the added benefit of studying HBx function at physiologic levels, thereby avoiding potential problems associated with HBx overexpression (e.g., aggregation, altered subcellular localization, etc.). Use of the empty plasmid vector is an appropriate negative control, depending on the experimental question. Overexpression of a control protein might be more appropriate for certain experiments such as those assessing effects of HBx on cellular stress, which could be affected by the overexpression of any protein and might not be specific to HBx. HBV plasmid replication assay. The HBV plasmid replication assay was a significant advance in the HBx field. In this assay, cells are transfected with plasmid DNA encoding a greater-than-unit length HBV genome, referred to as HBV 1.3mer (9), or with an identical plasmid containing mutations that prevent HBx expression (HBx-deficient HBV) (10, 11). Comparison of virus replication and gene expression between wildtype HBV and HBV plasmid that does not express HBx allows determination of the relative contribution of HBx. Importantly, the HBx-deficient phenotype is rescued by complementation with HBx provided by a second plasmid (12). Titration experiments showed that HBx levels below the limit of detection by immunoprecipitation/western blot assays were still sufficient to rescue HBx-deficient HBV replication (13). Similar results were obtained in mouse livers following hydrodynamic tail vein injection of the same plasmid DNA (14). Limitations and recommendations. The plasmid-based HBV replication assay is the staple for laboratories studying HBx in the context of HBV replication. . The effect of HBx on HBV replication is dependent on the cell type used for transfection. Significantly more virus replication is measured from the wildtype HBV plasmid (versus the HBx-deficient HBV plasmid) in HepG2 cells but not in Huh7 cells (15). Additionally, it is critical that cells be made quiescent (by increased plating density or by plating on collagen-treated plates) in order to reproducibly measure an effect of HBx on HBV replication (12, 14, 16). Finally, the magnitude of the HBx

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7 effect on HBV replication depends on the method used to quantify viral replication. HBxdeficient HBV replication can drop below the limit of detection by Southern blot, but can still be measured by real time PCR quantification of capsid-associated DNA (14). This can lead to differing interpretations of the contribution of HBx to HBV replication. The hydrodynamic injection model is a powerful in vivo model, but cannot assess early infection events (virus attachment, entry, etc.). The amount of plasmid DNA taken up by hepatocytes varies among individual injected animals. It is therefore essential to co-inject a plasmid DNA that expresses a reporter protein to monitor in vivo transfection efficiency among mice (14, 17). HBV infection models and HBx. The essential role of HBx in virus infection models was demonstrated in woodchucks (18, 19), human HepaRG cells (20), and human liver chimeric mice (21). In contrast, HBx is not absolutely required for virus replication driven from a transfected plasmid DNA or an integrated transgene. However, this HBx-independent HBV replication is enhanced by HBx expressed from a co-transfected plasmid (11). Limitations and recommendations. Most researchers do not have access to woodchucks, making accessibility of WHV experiments a limitation. However, collaborations with established WHV investigators may increase the feasibility of this approach. The HepaRG cell model has provided valuable information about HBx in the context of the entire virus life cycle, although only 10% of differentiated HepaRG cells are infected with HBV (22). Moreover, HBV cccDNA is not amplified in HepaRG cells, and assays measuring virus replication must be sensitive. The recent development of cell lines expressing the Sodium Taurocholate Cotransporting Polypeptide membrane protein that serves as an HBV receptor provides a new HBV infection system that will be useful in HBx research (23). These cells provide a routine and standardized HBV infection system in which to investigate HBx function(s). Despite the challenges of working with HBV-infection models, these assays are essential for understanding the role of HBx in the virus life cycle, particularly in light of the variability of HBx activities

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8 observed based on plasmid expression in different experimental systems. We recommend that one or more HBV infection models be used to confirm HBx effects observed in transient transfection assays. HBV transgenic mice can also be used, although there are limitations (see below). The role of HBx in HBV-associated carcinogenesis Several properties of HBx are consistent with a role in liver carcinogenesis [reviewed in (3, 24)]. Understanding the contribution(s) of HBx to the development of HBV-associated HCC has been hindered by the complexity of HBV pathogenesis that occurs over decades and includes integration of portions of HBV DNA into the host chromosome. Chronic HBV infection can be divided into an immune-active phase, during which HBV replicates to high levels, and an inactive carrier phase in which virus replication is curtailed (25). Both integrated HBV and host DNA are modified with deletions, insertions, and inversions (26). Characterization of HBVassociated HCCs by immunohistochemistry has shown that HBx can sometimes continue to be expressed even though HBV replication is not apparent (27, 28). Therefore, studies on HBx function(s) in tumors, and how these influence hepatocyte transformation, may sometimes allow that HBx effects be assessed in the absence of HBV replication. However, care should be taken to ensure that HBx levels in these studies mimic HBx levels in liver tumors or transformed cells. Mouse models of HBx oncogenic activities. HBx-transgenic mice have been created in a variety of genetic backgrounds, with HBx expression driven by viral or cellular promoters (Table 2). Most lineages of HBx-transgenic mice do not exhibit an abnormal pathological phenotype, but some do. The reason for these discrepant results cannot be explained by differences in the genetic background of the mice. In studies documenting HBx-induced HCC in HBx-transgenic mice, the authors noted the presence of additional confounding HCC risk factors (Table 2). However, there is general agreement that HBx acts as a cofactor in hepatocarcinogenesis in HBx-transgenic mice when combined with chemical carcinogens or activated oncogenes (Table

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9 2). HBV-transgenic mice with and without mutations that prevent HBx expression showed increased sensitivity to the carcinogen diethylnitrosamine (DEN) for the development of HCC; however, the sensitivity to DEN was higher in the presence of HBx (29). These results indicate that HBx, as well as other HBV-encoded proteins, contribute to DEN-mediated hepatocarcinogenesis (29). Limitations and recommendations. Transgenic mice are immune-tolerant to HBx (or HBV) and do not develop hepatitis or liver cirrhosis mediated by an immune response to HBV or HBx (30). In general, HBx expression or HBV replication does not cause liver pathology in these transgenic mice, although an increased incidence of HCC has been reported in older HBVtransgenic mice (29). Studies utilizing HBV-transgenic mice require careful planning. There may be variations in HBV protein expression or replication levels in different mice, and it is critical to select mice with similar serum levels of the secreted HBV e antigen, and to use at least two mice per group. Due to effects of age and gender on HBV gene expression (31, 32), age and sex-matched mice should always be used for paired studies. When possible, at least two independent lineages of HBV- or HBx-transgenic mice should be studied to rule out nonspecific effects of transgene integration. Use of nontransgenic control littermates is critical, unless there are scientific reasons to do otherwise. At a minimum, control mice should be housed similarly to transgenic mice to reduce the influence of environmental factors.

HBx expression levels in transgenic mice are also an important consideration. The use of strong, liver-specific promoters to drive HBx expression may lead to expression levels that are not physiologically relevant. While most studies with HBx-transgenic mice do not address this possibility, HBx expression in ATX mice was at levels very similar to that of WHx expressed during chronic WHV infection [discussed in (33)]. Furthermore, it is difficult to measure absolute HBx levels among different lineages of transgenic mice, in part because the available HBx antibodies may react variably to different subtypes of HBx [discussed in (14)].

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10 Cell lines used for studying HBx oncogenic activities. Most studies investigating HBx mechanisms in HCC have employed transformed liver cell lines such as HepG2 and Huh7 cells that are transfected with plasmid DNA encoding HBx. Many HBx functions have been proposed to influence HCC development [reviewed in (1, 3, 24)]. Limitations and recommendations. Cell lines often used to study HBx activities are tumorderived and already transformed. Consequently, the observed HBx-induced changes (in cellular protein or RNA levels, including noncoding RNAs such as miRNAs) may not be identifying direct events associated with the effect of HBx on transformation. Alternative cell culture systems for studying mechanisms of HBx-associated oncogenesis include primary rodent and human hepatocyte cultures and nontransformed immortalized liver cells e.g., THLE3, LO2, NeHepLxHT, or MIHA cells (34, 35). Importantly, Chang liver cells are contaminated with HeLa cells (36) and are inappropriate for studies on molecular mechanisms of HBx and liver cancer. Caution is needed when comparing HepG2 to HepG2.2.15 cells. HepG2 cells were established in 1979 from a pediatric hepatoblastoma (37), while the HepG2.2.15 cells are a clonal derivative of HepG2 cells and were established in 1987 following HBV plasmid transfection (38). Differences between these two cell lines are probably not solely due to HBV or HBx expression but perhaps to prolonged culture. Given the potential problems associated with overexpression of HBx, the level of HBx should be close to “physiologically relevant” levels in comparison to levels detected in chronic HBV infection or HCC tumors. It is possible to compare protein lysates of transfected HBxexpressing cells with protein lysates of HCC tissues on the same SDS-PAGE gel followed by immunoblot detection of HBx (35). Once normalized to a cellular protein loading control, a valid comparison can be made about HBx levels. The requirement to confirm HBx function using assays that mimic HBV replication may not always apply to studies of HBx function in carcinogenesis or in tumors since HBV replication is not always present in HBV-associated transformed hepatocytes and HCC.

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11 HBx-inducible cell lines. Hepatocyte cell lines can be difficult to transfect, and this has led to the use of strong promoters to express HBx, with the resulting problems associated with HBx overexpression. One alternative approach to regulate HBx expression levels in cells is through construction of stable, inducible expression systems. Several stable cell lines have been constructed in which inducible promoters regulate HBx expression. One example is the tetracycline (Tet)-off system constructed in the immortalized mouse AML12 cell line. HBx expression is induced by removal of tetracycline (39), allowing measurement of the immediate effects of HBx on the cell (40). The companion cells grown with tetracycline provide an appropriate negative control. The HepAD38 cell line harbors the whole HBV genome under control of the Tet-off system and exhibits tetracycline-regulated expression of pgRNA, the template of HBV replication (41). However, the mRNAs encoding PreS1, PreS2, and (presumably) HBx are expressed from their native promoters on the viral genome and are not regulated by tetracycline. The inducible

cells provide the context of virus replication and allow

study of temporal events involved in HBV replication. With stably integrated plasmid DNA present in each cell, these HBx- and HBV-inducible cell lines bypass difficulties associated with transfection of hepatocyte cell lines. Limitations and recommendations. While studies with HBx-inducible cell lines have identified cellular pathways altered by HBx (39, 42), these cell lines lack the context of the other viral proteins, which is important for establishing the relevance of the HBx function to the HBV life cycle (but not necessarily for studies on HBx function related to carcinogenesis). Conversely, the role of HBx in virus replication in HepAD38 cells, which contain the entire HBV genome, is not easily studied. A suggested solution is to confirm that events measured in cells stably expressing the whole HBV genome can be reproduced in inducible cell lines that express only HBx. This approach was recently used to show the role of HBx in epigenetic regulation (43, 44) and to demonstrate that HBV and HBx disrupt mitochondrial dynamics (6). A technical suggestion in the use of inducible systems is to exercise caution to avoid problems with

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12 “leakiness”. We recommend use of early passage of inducible cell lines and confirmation of inducible expression of genes of interest by RT-PCR or immunoblots. Carboxy-terminal deletion mutants of HBx. During HBV replication, portions of the HBV genome can be integrated into host chromosomal DNA. Although integration is thought to occur randomly, the region of the viral genome that spans the two direct repeats (DR) of HBV (Fig. 1) is especially proficient at recombination with cellular DNA (45, 46). Viral replication intermediates may provide the single-stranded DNA that invades the cellular DNA, leading to HBV integrations at this region of the viral genome (45). Regardless of the mechanism, there are two outcomes of this type of integration. First, the carboxyl portion of the X gene is deleted, with a possible effect on HBx function (47). Second, a viral enhancer upstream of the X gene (Fig. 1) may be brought into proximity of cellular genes that promote carcinogenesis. Limitations and recommendations. It is difficult to determine whether carboxyl-terminal HBx deletion mutants are drivers of carcinogenesis or if the tumor containing a carboxylterminal HBx deletion mutant is instead caused by HBV-genome integration that places a viral enhancer near cellular genes that promote carcinogenesis. Data needed to support the idea that truncated HBx drives tumor formation includes demonstration that HBx truncation mutant proteins are expressed in the tumor. Antibody staining of tumors will not distinguish between wildtype HBx and truncated HBx, and immunoblot analysis of tumor extracts may be difficult to interpret because of the possibility of HBx-host chimeric proteins co-migrating with wildtype HBx. Thus, there are significant challenges remaining in this area of research. At a minimum, it is necessary to demonstrate the presence of an intact HBx coding sequence by RNA sequencing, although interpretation of such data is complicated by the fact that the X ORF is in all HBV mRNAs (Fig. 1).

Other important considerations

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13 The following brief discussions highlight topics in the HBx field that are particularly relevant to investigators new to studying HBx. HBx is multifunctional. Considering the small size of the HBV genome, the virus relies heavily on the host cell to accomplish its life cycle (Fig. 2). Thus, it is not surprising that HBx is multifunctional [reviewed in (2, 3, 5). The proposed unstructured nature of the amino terminus of HBx (Fig. 3) supports its potential to interact with many cellular proteins (1). Some small viral regulatory proteins that are similarly unstructured can assemble and function as protein polymers with multiple functions in their virus life cycle (48, 49). Epitope tagged HBx. There are limited HBx antibodies, and investigators often use epitope-tagged versions of HBx for their studies. Although addition of these tags to the small HBx protein may alter its conformation and/or function, available evidence does not support this concern (13, 50). Nevertheless, there should be a continued effort to optimize and increase the number of commercially available HBx antibodies. Rigorous testing of these reagents in various applications is needed, and new HBx antibodies will serve as valuable tools for the entire HBx research community. Silencing RNA (siRNA) approaches and HBx research. Small interfering RNA (siRNA) technology provides a powerful tool for studying the function of genes. Knockdown of HBV replication has been achieved by siRNA indicating promising therapeutic applications [reviewed in (51)]. However, the siRNA approach should be used with caution in studies focusing on HBx function in model systems in which the entire HBV genome is present. Because the X gene is present in all HBV mRNAs (Fig. 1), any siRNA that targets the X mRNA may also target other HBV transcripts. Associated siRNA effects on HBV replication or cellular signal transduction pathways may not necessarily occur through an HBx-specific mechanism.

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14 Summary Understanding the role(s) of HBx in the HBV life cycle and in HBV-associated hepatocarcinogenesis remains a significant technical challenge due to limited reagents and technically difficult assays. Many assays investigate HBx function without providing a link to HBV replication, or under conditions of HBx expression levels that are unlikely to be encountered in HCCs. Given the short half-life of HBx and its potential unstructured region, there is concern for complications by HBx overexpression. It is imperative that studies of HBx functions in the HBV life cycle include approaches and assays in which HBx is expressed at biologically relevant levels and in the context of HBV replication. For studies of HBx activities in HCCs that could provide insights into its oncogenic functions, efforts should be made to ensure that the level of HBx is consistent with that observed in HCCs. Given that HBV replication and HBV infection models are now available, it is time for well-planned research that will reveal the role(s) of HBx in the HBV life cycle and HBV-associated HCC.

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15 Figure Legends

Figure 1: Organization of the HBV genome. The HBV genome is a partially double-stranded DNA. The central circle depicts the conventional numbering of the circular genome where positions 1 and 3182 usually represent a unique EcoR1 restriction site. Small differences in genome length among genotypes (and serotypes) exist; the ayw serotype is shown. The black circular lines represent the DNA genome with a completed negative-strand DNA (-strand) and a partially completed (dashed lines) positive-strand DNA (+ strand). Also indicated are: direct repeats (DR1, DR2), enhancers (EN1, EN2) and polymerase (orange circle labeled "pol"). Colored arrows indicate the ORFs for preCore, core, polymerase, envelope (preS1, pres2, and S), and HBx proteins. Outer black arrows depict genomic and subgenomic polyadenylated transcripts transcribed from cccDNA, also depicted at the bottom of the figure highlighting overlapping regions. Colored boxes below the circular genome indicate ORFs for HBV proteins, highlighting their overlapping regions. Figure 2: The HBV lifecycle. HBV infects susceptible cells by interacting with its cell-surface receptor, the sodium taurocholate cotransporting polypeptide (NTCP), also known as SLC10A1. HBV enters the cell by an incompletely understood process, loses its envelope, and the capsidenclosed genome is transported to the nucleus and releases the genome into the nucleus. In the nucleus, the partially double-stranded DNA genome (dsDNA) is repaired and converted to covalently closed, circular DNA (cccDNA), which is the template for HBV transcripts. HBV transcripts are transported to the cytoplasm and translated to produce HBV proteins. One of the transcripts, the pregenomic RNA is encapsidated and reverse-transcribed to the HBV DNA genome. Envelopment of genome-containing capsids occurs, and HBV is then released from the cell. Some of the encapsidated genome does not acquire an envelope and can be recycled back to the nucleus to contribute to the pool of nuclear cccDNA.

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16 Figure 3: Schematic of the HBx protein (box). HBx is a 154 amino acid, 17kD protein. Lines below the HBx schematic represent identified functional regions of HBx. A region that inhibits some HBx activities is located at the N-terminus of HBx (Negative). Regions required for binding to transcription factors (Transcription factor binding) and Damage DNA Binding Protein 1 (DDB1 binding) are indicated. A nuclear export signal (nuclear export) is also indicated. Comparative sequence analysis of HBx from multiple genotypes has identified a hypervariable region (Hypervariable). Asterisks (*) indicate conserved cysteines.

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17 Acknowledgements The authors thank Dr. Jason Lamontagne for preparation of Figure 1, and Drs. Joseph Hyser and Robert J Schneider for their critical review of the manuscript. Betty Slagle served as lead author. The co-authors are listed in alphabetical order.

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20 28. Poussin K, Dienes H, Sirma H, Urban S, Beaugrand M, Franco D, et al. Expression of mutated hepatitis B virus X genes in human hepatocellular carcinomas. Int J Cancer 1999;80:497-505. 29. Zheng Y, Chen WL, Louie SG, Yen TS, Ou JH. Hepatitis B virus promotes hepatocarcinogenesis in transgenic mice. Hepatology 2007;45(1):16-21. 30. Guidotti LG, Chisari FV. Immunobiology and pathogenesis of viral hepatitis. Annu Rev Pathol 2006;1:23-61. 31. Tian Y, Kuo CF, Chen WL, Ou JH. Enhancement of hepatitis B virus replication by androgen and its receptor in mice. J VIrol 2012;86(4):1904-1910. 32. Wang SH, Yeh SH, Lin WH, Yeh KH, Yuan Q, Xia NS, et al. Estrogen receptor alpha represses transcription of HBV genes via interaction with hepatocyte nuclear factor 4alpha. Gastroenterology 2012;142(4):989-998. 33. Madden CR, Finegold MJ, Slagle BL. Hepatitis B virus X protein acts as a tumor promoter in the development of diethylnitrosamine-induced preneoplastic lesions. J Virol 2001;75:3851-3858. 34. Gao Y, Theng SS, Zhuo J, Teo WB, Ren J, Lee CG. FAT10, an ubiquitin-like protein, confers malignant properties in non-tumorigenic and tumorigenic cells. Carcinogenesis 2014;35(4):923-934. 35. Chan C, Wang Y, Chow PK, Chung AY, Ooi LL, Lee CG. Altered binding site selection of p53 transcription cassettes by hepatitis B virus X protein. Mol Cell Biol 2013;33(3):485497. 36. Nelson-Rees WA, Flandermeyer RR. HeLa cultures defined. Science 1976;191(4222):9698. 37. Aden DP, Fogel A, Plotkin S, Damjanov I, Knowles BB. Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line. Nature 1979;282:615-616. 38. Sells MA, Chen M-L, Acs G. Production of hepatitis B virus particles in HepG2 cells transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci 1987;84:1005-1009. 39. Tarn C, Bilodeau ML, Hullinger RL, Andrisani OM. Differential immediate early gene expression in conditional hepatitis B virus pX-transforming versus nontransforming hepatocyte cell lines. J Biol Chem 1999;274:2327-2336. 40. Tarn C, Lee S, Hu Y, Ashendel C, Andrisani OM. Hepatitis B virus X protein differentially activates RAS-RAF-MAPK and JNK pathways in X-transforming versus non-transforming AML12 hepatocytes. J Biol Chem 2001;276(37):34671-34680. 41. Ladner SK, Otto MJ, Barker CS, Zaifert K, Wang GH, Guo JT, et al. Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: A novel system for screening potential inhibitors of HBV replication. Antimicrob Agents Chemother 1997;41:1715-1720.

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21 42. Andrisani OM. Deregulation of epigenetic mechanisms by the hepatitis B virus X protein in hepatocarcinogenesis. Viruses 2013;5(3):858-872. 43. Wang WH, Studach LL, Andrisani OM. Proteins ZNF198 and SUZ12 are down-regulated in hepatitis B virus (HBV) X protein-mediated hepatocyte transformation and in HBV replication. Hepatol 2011;53(4):1137-1147. 44. Studach LL, Menne S, Cairo S, Buendia MA, Hullinger RL, Lefrancois L, et al. Subset of Suz12/PRC2 target genes is activated during hepatitis B virus replication and liver carcinogenesis associated with HBV X protein. Hepatology 2012;56(4):1240-1251. 45. Nagaya T, Nakamura T, Tokino T, Tsurimoto T, Imai M, Mayumi T, et al. The mode of hepatitis B virus DNA integration in chromosomes of human hepatocellular carcinoma. Genes Dev 1987;1(8):773-782. 46. Paterlini-Brechot P, Saigo K, Murakami Y, Chami M, Gozuacik D, Mugnier C, et al. Hepatitis B virus-related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets human telomerase gene. Oncogene 2003;22(25):3911-3916. 47. Sirma H, Giannini C, Poussin K, Paterlini P, Kremsdorf D, Bréchot C. Hepatitis B virus X mutants, present in hepatocellular carcinoma tissue abrogate both the antiproliferative and transactivation effects of HBx. Oncogene 1999;18:4848-4859. 48. Ou HD, Kwiatkowski W, Deerinck TJ, Noske A, Blain KY, Land HS, et al. A structural basis for the assembly and functions of a viral polymer that inactivates multiple tumor suppressors. Cell 2012;151(2):304-319. 49. Bornholdt ZA, Noda T, Abelson DM, Halfmann P, Wood MR, Kawaoka Y, et al. Structural rearrangement of Ebola virus VP40 begets multiple functions in the virus life cycle. Cell 2013;154(4):763-774. 50. McClain SL, Clippinger AJ, Lizzano R, Bouchard MJ. Hepatitis B virus replication is associated with an HBx-dependent mitrochondrion-regulated increase in cytosolic calcium levels. J Virol 2007;81:12061-12065. 51. Arbuthnot P, Longshaw V, Naidoo T, Weinberg MS. Opportunities for treating chronic hepatitis B and C virus infection using RNA interference. J Viral Hepat 2007;14(7):447459.

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Table 1. Models for the Study of HBV Replication and HBx1 HBV replication step Model Entry Human HepG2 + HBV 1.3mer HepAd38 HepaRG hNTCP HepG2 Cultured primary hepatocytes Human liver chimera Mouse or rat Hydrodynamic injection of mice with HBV 1.3mer Cultured rat primary hepatocytes HBV transgenic mice

ccc DNA

4

2

HBx-dependent

Assembly

Egress

Viremia

Adaptive Immunity

NA NA + + +

+ + + + +

5

+ + + + +

+ + + + +

NA NA NA NA NA

NA NA NA NA NA

Yes/No 7 ND Yes ND Yes

+

ND

+

+

+

NA

Yes

NA

-

8

+

+

+

+

Yes/No

ND

ND

+

+

NA

NA

ND

NA

-

9

+

+

+

10

Yes/No

-

3

6

1

See text for references to models listed.

2

Step in the HBV life cycle: Entry, attachment and entry of virus; cccDNA, presence of cccDNA template; Assembly, viral proteins form capsids and particles; Egress, virus particles bud from the cell; Viremia, detection of virus in serum; Adaptive immunity, presence of adaptive immune response against HBV.

3

HBV replication in this model is dependent on HBx

4

NA, not applicable; this step in virus life cycle cannot be measured in this model.

5

”+“, step in virus replication can be assessed in this model and is found

6

Yes/No, virus replication can be measured in the absence of HBx but is increased in the presence of HBx.

7

ND, has not been determined for this model

8 “-“

, Step in virus replication has been assessed and is not found in this model

9

cccDNA not detected, except as reported in in J. Virol. 75:2900-2911, 2001.

10

Can be assessed using adoptively transferred immune cells from HBV-antigen exposed, nontransgenic syngeneic mice.

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Table 2. HBx Transgenic Mice1 Regulatory Genetic Background3 Region2

Pathology observed (% of total mice)

Role of HBx in HCC Reference

Untreated mice (spontaneous HCC) ATX HBV

ICR (Outbred) None CD-1 (Outbred) HCC (100%)

None 4 Cofactor

Lee et al (1990) Kim et al (1991)

HBV

C57BL6/6J

None

None

Reifenberg et al (1997)

INS

C57BL6/6J

None

None

Reifenberg et al 1997)

PEX7

C57BL6/6

None

None

Billet et al (1995)

SVX

C57BL6/6

None

None

Billet et al (1995)

AX16

C57BL6/6

None

None

Billet et al (1995)

WAP

NMRI (inbred)

None

None

Klein et al (2003)

Cofactor

5

Yu et al (1999)

ALB C57BL/6 HCC (86%) Mice treated with cancer cofactor

Cofactor

6

Wu et al (2006)

ATX

ICR (Outbred)

HCC with DEN

Cofactor

Slagle et al (1996)

WHV

Cofactor

Dandri et al (1996)

HBV

C57BL/6

HCC (86%)

HBV

CD-1 (Outbred) HCC with DEN C57BL/6/DBA2 HCC with *myc

Cofactor

Terradillos et al (1997)

AX16

C57BL/6/DBA2

HCC with *myc

Cofactor

Terradillos et al (1997)

ATX

ICR (outbred)

HCC with HCV

Cofactor

Keasler et al (2006)

WAP

NMRI (albino)

Cofactor

Klein et al ((2003)

HBV

C57BL6

Mammary carcinoma in p53 +/- mice HCC with p21 knockout

Cofactor

Wang et al (2004)

HBV

C57BL6

HCC with DEN

Cofactor

Zhu et al (2004)

HBV

C57BL6

HCC with DDC diet

Cofactor

Wang et al (2012)

1

Table modified with permission from Hodgson AJ and Slagle BL. Molecular Biology of HBV-related Hepatocellular Carcinoma. In: Shih C., ed. Chronic Hepatitis B and C. Singapore: World Scientific Publishing Co. Pte. Ltd, 2012. 99-131.. 2 Regulatory regions used to drive HBx expression included ATX (human alpha-1-antitrypsin), HBV (native HBx promoter/enhancer), INS (rat insulin), ALB (albumin), Woodchuck hepatitis B virus promoter; WAP, whey acidic protein). 3 Genetic background of the mice used to generate transgenics 4 Spontaneous HCCs were reported in nontransgenic controls 5 Diagnosis of HCC corrected to biliary cysts by Dirsch et al (2004) 6 Accumulation of hepatocyte fat (steatosis) may have contributed to HCCs

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170x142mm (300 x 300 DPI)

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254x190mm (96 x 96 DPI)

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127x43mm (300 x 300 DPI)

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