**2. Hepatitis B virus**

HBV is a member of the *Hepadnaviridae,* a family of hepatotropic viruses that predominately infect hepatocytes in their respective hosts; similar viruses have been isolated from apes, woodchucks, squirrels, ducks, geese, and cranes [reviewed in (Seeger et al, 2007)]. HBV has a highly compact DNA genome of about 3200 nucleotides in length that contains four overlapping open reading frames (ORFs); every nucleotide of the genome is in at least one

<sup>\*</sup> Co-first authors

Modulation of Cell Proliferation Pathways by the Hepatitis B

on hepatocyte physiology (Gearhart & Bouchard, 2011).

detailed description.

Virus X Protein: A Potential Contributor to the Development of Hepatocellular Carcinoma 105

the limited availability of cultured primary human hepatocytes, has lead many researchers to study HBV replication and the activities of HBV-encoded proteins in immortalized or transformed liver cell lines and in cultured primary hepatocytes derived from small animal models such as rats or mice [reviewed in (Bouchard & Navas-Martin, 2011; Seeger et al, 2007)]. Use of these systems necessitates the bypass of the initial receptor-mediated infection of the cell by direct transfection of the HBV DNA genome. Although studies in immortalized or transformed cells have served as powerful models for studying various aspects of HBV replication and the functions of HBV-encoded proteins, these studies have also demonstrated that the activities of HBV proteins may vary in different cellular contexts [reviewed in (Bouchard & Navas-Martin, 2011; Neuveut et al, 2010)]. Studies in cultured primary hepatocytes have begun to clarify HBV replication strategies and the function of HBV proteins in a more relevant context [reviewed in (Bouchard & Navas-Martin, 2011)]. Recently, cultured primary rat hepatocytes have been used to study HBV replication and functions of the HBx protein (Clippinger & Bouchard, 2008; Clippinger et al, 2009; Gearhart & Bouchard, 2010a; Gearhart & Bouchard, 2010b); HBx activities in cultured primary rat hepatocytes were similar to HBx activities in cultured primary human hepatocytes, supporting the use of cultured primary rat hepatocytes as a good model system for studying the impact of HBV

Fig. 1. Schematic depiction of the HBV genome. HBV DNA is denoted by bold lines with black arrows, HBV open reading frames (ORFs) are represented by inner arrows shown in gray, and HBV mRNAs are represented by outer arrows shown in white. See text for

open reading frame (Figure 1). The four overlapping ORFs of the HBV genome encode seven proteins: the reverse transcriptase/polymerase, the pre-core (E antigen) and core (capsid) proteins, three envelope (large, middle, and small hepatitis B surface antigens) proteins, and the nonstructural X protein (HBx) [reviewed in (Seeger et al, 2007)]. The cellsurface receptor of HBV that facilitates HBV infection of hepatocytes has not been conclusively identified, and the mechanism by which HBV enters hepatocytes is not clear. Upon infection of hepatocytes, the encapsidated, partially double-stranded DNA genome is transported to the nucleus, where it is converted into a covalently closed, double-stranded, circular DNA (cccDNA). cccDNA is the template for all HBV RNA transcripts; the transcripts are exported out of the nucleus into the cytoplasm and are translated to form the HBV pre-core, core, envelope, reverse transcriptase/polymerase, and HBx proteins. The largest HBV RNA transcript, the pregenomic RNA (pgRNA), is packaged with the reverse transcriptase into viral capsids in the cytosol and is reverse transcribed to generate the partially double-stranded HBV DNA genome [reviewed in (Seeger et al, 2007)]. HBV viral capsids containing the replicating genome bud into the endoplasmic reticulum by envelopment within the HBV envelope proteins and are secreted from the infected hepatocyte [reviewed in (Nguyen et al, 2008; Seeger et al, 2007)].

#### **2.1 Model systems for studying HBV infections**

Each member of the hepadnavirus family has a narrow host range that is thought to be defined primarily by the interaction between the virus and a specific receptor that is present on the surface of host hepatocytes [reviewed in (Seeger et al, 2007)]. Available cell culture systems for studying the life cycle of the *Hepadnaviridae* are limited. Typically, members of the hepadnavirus family can only directly infect hepatocytes within the liver of their respective avian or mammalian hosts or cultured, well-differentiated primary hepatocytes that are derived from these hosts; this has hampered the capabilities of researchers to study a natural HBV infection [reviewed in (Koike, 2009; Seeger et al, 2007)]. Chimpanzees are the most relevant animal model for studying the consequences of an HBV infection; however, due to cost and ethical reasons, studies in chimpanzees are limited (Prince & Brotman, 2001). HBV infections in chimpanzees also do not completely mimic all aspects of an HBV infection in humans; chimpanzees chronically infected with HBV usually do not develop liver cirrhosis or HCC (Prince & Brotman, 2001). HBV-transgenic mice have served as an important small animal model for studying *in vivo* HBV replication and immune-mediated HBV clearance from hepatocytes [(Guidotti et al, 1995); reviewed in (Guidotti & Chisari, 2006)]. However, because HBV-transgenic mice contain a copy of the HBV genome that is integrated into the genome of all hepatocytes in these mice, the consequences of chronic inflammation for the development of HBV-associated HCC cannot be studied in this model [reviewed in (Bouchard & Navas-Martin, 2011)]. More recently, human hepatocyte chimeric mice, which were generated by the replacement of the majority of the mouse hepatocytes with implanted human hepatocytes, have been used to study HBV infections in what may become a more experimentally tractable and relevant model than other currently available small animal model systems (Tsuge et al, 2005). Mice with humanized-livers that were inoculated with HBV had high levels of HBV viremia that lasted for up to 22 weeks (Tsuge et al, 2005). Hydrodynamic transfection of the HBV genome into mouse livers has also been used as a method for studying HBV replication in hepatocytes; however, due to rapid clearance of the virus, persistent HBV infection cannot be studied in this system (Keasler et al, 2007). The paucity of *in vivo* models for studying direct HBV infections, and

open reading frame (Figure 1). The four overlapping ORFs of the HBV genome encode seven proteins: the reverse transcriptase/polymerase, the pre-core (E antigen) and core (capsid) proteins, three envelope (large, middle, and small hepatitis B surface antigens) proteins, and the nonstructural X protein (HBx) [reviewed in (Seeger et al, 2007)]. The cellsurface receptor of HBV that facilitates HBV infection of hepatocytes has not been conclusively identified, and the mechanism by which HBV enters hepatocytes is not clear. Upon infection of hepatocytes, the encapsidated, partially double-stranded DNA genome is transported to the nucleus, where it is converted into a covalently closed, double-stranded, circular DNA (cccDNA). cccDNA is the template for all HBV RNA transcripts; the transcripts are exported out of the nucleus into the cytoplasm and are translated to form the HBV pre-core, core, envelope, reverse transcriptase/polymerase, and HBx proteins. The largest HBV RNA transcript, the pregenomic RNA (pgRNA), is packaged with the reverse transcriptase into viral capsids in the cytosol and is reverse transcribed to generate the partially double-stranded HBV DNA genome [reviewed in (Seeger et al, 2007)]. HBV viral capsids containing the replicating genome bud into the endoplasmic reticulum by envelopment within the HBV envelope proteins and are secreted from the infected

Each member of the hepadnavirus family has a narrow host range that is thought to be defined primarily by the interaction between the virus and a specific receptor that is present on the surface of host hepatocytes [reviewed in (Seeger et al, 2007)]. Available cell culture systems for studying the life cycle of the *Hepadnaviridae* are limited. Typically, members of the hepadnavirus family can only directly infect hepatocytes within the liver of their respective avian or mammalian hosts or cultured, well-differentiated primary hepatocytes that are derived from these hosts; this has hampered the capabilities of researchers to study a natural HBV infection [reviewed in (Koike, 2009; Seeger et al, 2007)]. Chimpanzees are the most relevant animal model for studying the consequences of an HBV infection; however, due to cost and ethical reasons, studies in chimpanzees are limited (Prince & Brotman, 2001). HBV infections in chimpanzees also do not completely mimic all aspects of an HBV infection in humans; chimpanzees chronically infected with HBV usually do not develop liver cirrhosis or HCC (Prince & Brotman, 2001). HBV-transgenic mice have served as an important small animal model for studying *in vivo* HBV replication and immune-mediated HBV clearance from hepatocytes [(Guidotti et al, 1995); reviewed in (Guidotti & Chisari, 2006)]. However, because HBV-transgenic mice contain a copy of the HBV genome that is integrated into the genome of all hepatocytes in these mice, the consequences of chronic inflammation for the development of HBV-associated HCC cannot be studied in this model [reviewed in (Bouchard & Navas-Martin, 2011)]. More recently, human hepatocyte chimeric mice, which were generated by the replacement of the majority of the mouse hepatocytes with implanted human hepatocytes, have been used to study HBV infections in what may become a more experimentally tractable and relevant model than other currently available small animal model systems (Tsuge et al, 2005). Mice with humanized-livers that were inoculated with HBV had high levels of HBV viremia that lasted for up to 22 weeks (Tsuge et al, 2005). Hydrodynamic transfection of the HBV genome into mouse livers has also been used as a method for studying HBV replication in hepatocytes; however, due to rapid clearance of the virus, persistent HBV infection cannot be studied in this system (Keasler et al, 2007). The paucity of *in vivo* models for studying direct HBV infections, and

hepatocyte [reviewed in (Nguyen et al, 2008; Seeger et al, 2007)].

**2.1 Model systems for studying HBV infections** 

the limited availability of cultured primary human hepatocytes, has lead many researchers to study HBV replication and the activities of HBV-encoded proteins in immortalized or transformed liver cell lines and in cultured primary hepatocytes derived from small animal models such as rats or mice [reviewed in (Bouchard & Navas-Martin, 2011; Seeger et al, 2007)]. Use of these systems necessitates the bypass of the initial receptor-mediated infection of the cell by direct transfection of the HBV DNA genome. Although studies in immortalized or transformed cells have served as powerful models for studying various aspects of HBV replication and the functions of HBV-encoded proteins, these studies have also demonstrated that the activities of HBV proteins may vary in different cellular contexts [reviewed in (Bouchard & Navas-Martin, 2011; Neuveut et al, 2010)]. Studies in cultured primary hepatocytes have begun to clarify HBV replication strategies and the function of HBV proteins in a more relevant context [reviewed in (Bouchard & Navas-Martin, 2011)]. Recently, cultured primary rat hepatocytes have been used to study HBV replication and functions of the HBx protein (Clippinger & Bouchard, 2008; Clippinger et al, 2009; Gearhart & Bouchard, 2010a; Gearhart & Bouchard, 2010b); HBx activities in cultured primary rat hepatocytes were similar to HBx activities in cultured primary human hepatocytes, supporting the use of cultured primary rat hepatocytes as a good model system for studying the impact of HBV on hepatocyte physiology (Gearhart & Bouchard, 2011).

Fig. 1. Schematic depiction of the HBV genome. HBV DNA is denoted by bold lines with black arrows, HBV open reading frames (ORFs) are represented by inner arrows shown in gray, and HBV mRNAs are represented by outer arrows shown in white. See text for detailed description.

Modulation of Cell Proliferation Pathways by the Hepatitis B

**3. HBx** 

references and details.

multiple mechanisms are likely to influence HCC development.

Virus X Protein: A Potential Contributor to the Development of Hepatocellular Carcinoma 107

that could influence HBV replication, hepatocyte transformation, and HCC development [reviewed in (Benhenda et al, 2009; Koike, 2009; Neuveut et al, 2010)]. Although this chapter focuses on HBx activities, it is important to note that in the context of an HBV infection,

HBx is a 154 amino acid, 17 kDa, multifunctional protein that is encoded by the smallest open reading frame of the HBV genome. HBx can stimulate HBV replication and modulate intracellular calcium signaling, cell proliferation and apoptotic pathways, signal transduction pathways, and the activity of various transcription factors and the proteasome (Figure 2) [reviewed in (Benhenda et al, 2009; Bouchard & Schneider, 2004; Koike, 2009)]. Because HBx activities appear to be influenced by the cell type and the method of HBx expression used in a study, not all HBx functions in normal hepatocytes and in the context of HBV replication are completely understood. Most studies that have assessed the effects of HBx have been performed in immortalized or transformed cells, when HBx was expressed at higher levels than observed during HBV replication, and when HBx was expressed outside of the context of HBV replication. Because HBx is expressed at low levels during HBV replication, it has been difficult to establish systems in which HBx interacting partners or HBx activities can be easily studied in the context of HBV replication. Consequently, the use of various cellular model systems to study HBx has sometimes identified seemingly discrepant HBx activities. It is important to note however, that many studies that have analyzed HBx activities in systems where HBx is expressed in the context of HBV replication and in normal hepatocytes have confirmed HBx activities that were identified when HBx

Fig. 2. Schematic representation of HBx activities. HBx is a multifunctional protein known to regulate HBV replication, numerous signal transduction pathways, the cell cycle, apoptosis, and transcription. HBx can also bind multiple cellular proteins. One or more of these HBx activities could contribute to the development of HBV-associated HCC. See text for

#### **2.2 HBV and hepatocellular carcinoma**

Common modes of HBV transmission include exposure to HBV-contaminated blood, unsafe injection practices, sexual contact with an HBV-infected individual, and perinatal transmission from an HBV-infected mother to her child (WHO, 2008)**.** HBV infections are classified as acute, chronic, or occult. In most people with acute or short-term HBV infections, the infection is resolved by immune-mediated viral clearance. The inflammatory response to the HBV infection causes many of the symptoms that have been associated with hepatitis; these symptoms include jaundice, fatigue, nausea, vomiting, and abdominal pain [(WHO, 2008); reviewed in (Seeger et al, 2007)]. Chronic HBV infections are characterized by the continued presence of detectable levels of the HBV small envelope protein in the blood of an HBV-infected individual for more than six months [reviewed in (Lok & McMahon, 2001)]. Chronic infections with either HBV or the hepatitis C virus (HCV) are estimated to account for more than 80% of primary liver cancers; approximately 60% of these liver cancers are attributed to chronic HBV infections and 40% to chronic HCV infections [reviewed in (El-Serag & Rudolph, 2007; Koike, 2009)]. Currently, the geographic locations with the highest incidence of HCC are areas where HBV infection is highly endemic [reviewed in (Pang et al, 2006)]. There are eight known genotypes of HBV, and mounting evidence suggests that increased persistence of an HBV infection and a greater risk for HCC development may be influenced by the HBV genotype in the infected individual [reviewed in (Chemin & Zoulim, 2009)]. Environmental factors such as alcohol consumption, aflatoxin exposure, and tobacco use increase the risk for developing HCC in HBV-infected individuals [reviewed in (Chemin & Zoulim, 2009)].

Although the association between chronic HBV infections and HCC development is clear, the mechanisms that link a chronic HBV infection to HCC development are incompletely understood. Three potential mechanisms have been commonly invoked as consequences of an HBV infection that could contribute to HCC development. The first mechanism that may contribute to the development of HBV-associated HCC is persistent liver inflammation and hepatocyte proliferation that is caused by recurrent immune-mediated destruction of HBVinfected hepatocytes and concomitant liver regeneration in chronically HBV-infected individuals [reviewed in (Bouchard & Navas-Martin, 2011; Guidotti & Chisari, 2006)]. Persistent liver inflammation can cause fibrosis and cirrhosis and may eventually select for hepatocytes that have accumulated tumorigenic properties [reviewed in (Bouchard & Navas-Martin, 2011; Chemin & Zoulim, 2009)]. Chronic liver inflammation, the associated elevation of reactive oxygen species (ROS), and the potential for ROS to cause DNA damage may also produce a more cancer-prone environment [reviewed in (Chemin & Zoulim, 2009)]. The second mechanism that has been proposed to contribute to the development of HBV-associated HCC is the possible consequence of HBV genome integration into the host genome, which could cause genetic or epigenetic changes and genomic instability [reviewed in (Chemin & Zoulim, 2009; Neuveut et al, 2010)]. Integration of the HBV genome into the host genome could potentially result in the loss of tumor suppressive functions and/or the gain of tumor-promoting activities [reviewed in (Chemin & Zoulim, 2009)]. The third mechanism that has been proposed to be involved in the development of HBV-associated HCC, and the primary focus of this chapter, is the alteration of hepatocyte physiology and stimulation of HBV replication that is linked to expression of the HBV HBx protein [reviewed in (Bouchard & Navas-Martin, 2011; Koike, 2009; Neuveut et al, 2010)]. The multifunctional HBx protein has been shown to affect numerous cellular signaling pathways that could influence HBV replication, hepatocyte transformation, and HCC development [reviewed in (Benhenda et al, 2009; Koike, 2009; Neuveut et al, 2010)]. Although this chapter focuses on HBx activities, it is important to note that in the context of an HBV infection, multiple mechanisms are likely to influence HCC development.
