**3. HBx**

106 Hepatocellular Carcinoma – Basic Research

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

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

**2.2 HBV and hepatocellular carcinoma** 

individuals [reviewed in (Chemin & Zoulim, 2009)].

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 references and details.

Modulation of Cell Proliferation Pathways by the Hepatitis B

**3.2 HBx and hepatocellular carcinoma** 

Virus X Protein: A Potential Contributor to the Development of Hepatocellular Carcinoma 109

with wild-type HBV (Tsuge et al, 2010; Tsuge et al, 2005). HBx expression also stimulates HBV replication in HepG2 cells, a human hepatoblastoma cell line, and in cultured primary rat hepatocytes [reviewed in (Bouchard & Schneider, 2004)]. Although most studies indicate that HBV replication in Huh7 cells, a human hepatoma cell line, is not regulated by HBx expression, a recent study demonstrated that HBx can also stimulate HBV replication in Huh7 cells (Lim et al, 2010). Many of the signaling pathways that are modulated by HBx, and will be discussed in the following sections of this chapter, have also been shown to influence HBV replication. HBx modulation of intracellular calcium signaling, activation of the Proline-rich tyrosine kinase 2 (Pyk2)/Focal adhesion kinase (FAK)-Src-Ras-Raf-MAPK signaling pathway, regulation of transcription pathways, interaction with ultraviolet DNA damage binding protein 1 (UVDDB1), and association with proteasome factors affect HBx regulation of HBV replication (Bouchard et al, 2001b; Gearhart & Bouchard, 2010b; Klein et al, 1999; Leupin et al, 2005; Tan et al, 2009; Tang et al, 2005; Zhang et al, 2004). Overall, studies in various model systems suggest that the X proteins of mammalian hepadnaviruses have an important stimulatory role during replication of mammalian hepadnaviruses.

HBx expression and activities are thought to be major contributing factors in the development of HBV-associated HCC; however, the exact contribution of HBx to HCC development is unknown. One important clue that implicates HBx in the development of HBV-associated HCC is that chronic infections of birds with avian hepadnaviruses are not associated with the development of HCC [reviewed in (Seeger et al, 2007)]. The avian hepadnaviruses either do not encode an X protein or encode an X protein that is highly divergent from the X proteins of mammalian hepadnaviruses (Mandart et al, 1984; Sprengel et al, 1988). The mammalian hepadnaviruses all encode an X protein, and only infections with mammalian hepadnaviruses are associated with the development of HCC in their respective hosts [reviewed in (Seeger et al, 2007)]. Additional evidence for the involvement of HBx in HCC development has come from studies in HBx-transgenic mice. Some studies have shown that HBx expression can directly cause HCC in HBx-transgenic mice (Kim et al, 1991; Yu et al, 1999). In contrast, other researchers have found that HBx expression alone is not sufficient for the development of liver tumors in HBx-transgenic mice but can sensitize these mice to chemical- or oncogene-induced HCC (Madden et al, 2001; Slagle et al, 1996; Terradillos et al, 1997; Zhu et al, 2004). While the reason for the observed differences in tumor development in the various HBx-transgenic mice has not been completely explored and may be related to the genetic background of the mice and/or the level of HBx expression, studies in these HBx-transgenic mice strongly support the notion that HBx can have at least a co-factor role in the development of HCC. Importantly, a co-factor role for HBx in the development of HBV-associated HCC is more consistent with the biology of HCC development in chronically HBV-infected individuals; development of HCC in chronically HBV-infected individuals can take decades to arise [reviewed in (Seeger et al, 2007)]. The observation that HBV-associated HCC requires decades to arise suggests that HBV does not encode a strongly oncogenic protein but instead encodes proteins that can cause subtle changes to hepatocyte physiology that could sensitize hepatocytes to other oncogenic signals [reviewed in (Bouchard & Navas-Martin, 2011; Neuveut et al, 2010)]. The

activities of HBx that could influence HCC development are discussed below.

was overexpressed out of the context of HBV replication, suggesting that studies in various cellular systems can provide valuable insights into HBx activities [reviewed in (Benhenda et al, 2009; Bouchard & Schneider, 2004; Koike, 2009)]. Moreover, because HBx is often expressed in HBV-associated liver tumor cells even when expression of other HBV proteins is not detectable (Wang et al, 2004b; Wollersheim et al, 1988), studies in immortalized or transformed cells may help identify HBx activities that could be present in transformed cells but absent in normal hepatocytes. Overall, it is likely that studies in immortalized or transformed cells, in cultured primary hepatocytes, in the livers of available animal models, and when HBx is expressed in the absence of other HBV proteins and in the context of HBV replication each contribute important information regarding the various activities of this protein. Considering the compact nature of the HBV genome, the limited number of proteins encoded by the HBV genome, and the necessity for these proteins to perform their functions in an environment that may not favor viral replication, it is not surprising that HBV proteins such as HBx, which is the only regulatory protein encoded by the HBV genome, have multiple functions that impact HBV replication and cellular physiology.

#### **3.1 HBx and HBV replication**

Many studies have indicated that replication of mammalian hepadnaviruses is stimulated by their respective X proteins. Two different studies performed in woodchucks showed that the woodchuck hepatitis virus (WHV) X protein (WHx) is absolutely required for WHV replication in the livers of woodchucks (Chen et al, 1993; Zoulim et al, 1994); however, in one dissenting study, another group detected a very low level of WHV replication in woodchucks infected with an WHx-deficient WHV mutant (Zhang et al, 2001). Evidence that HBx stimulates HBV replication has been generated from studies in various mouse models and cell culture systems. Transgenic mice were generated with either a wild-type HBV genome or a mutant form of the HBV genome that did not express HBx; HBV replication was detected in both the wildtype HBV and mutant HBV-transgenic mice (Xu et al, 2002). However, when the HBx-deficient HBV-transgenic mice were bred with HBxtransgenic mice, higher levels of circulating HBV viremia and higher levels of HBV core and envelope proteins were detected in the double transgenic mice than in their HBx-deficient, HBV-transgenic counterparts (Xu et al, 2002). These studies suggest that although HBx may not be absolutely required for HBV replication in this model system, HBx does enhance HBV replication in the livers of these mice. An important caveat to the studies in HBVtransgenic mice is that these mice do not produce nuclear HBV cccDNA and may not recapitulate all aspects of authentic HBV replication (Guidotti et al, 1995). Additional evidence that HBx stimulates HBV replication has come from a study where mice were hydrodynamically injected with a plasmid encoding the wild-type HBV genome or a mutant HBx-deficient HBV genome (Keasler et al, 2007). The results of these studies demonstrated that although HBx was not absolutely required for HBV replication, there was a very significant decrease in HBV replication in mice injected with the HBx-deficient HBV as compared to mice injected with the wildtype HBV genome (Keasler et al, 2007). Interestingly, when a plasmid encoding HBx was co-injected with a plasmid encoding the mutant HBx-deficient HBV genome, HBV replication levels were restored to those seen in mice injected with the plasmid encoding wild-type HBV (Keasler et al, 2007). Finally, studies in mice with humanized livers showed that after direct infection with wild-type HBV or an HBx-deficient HBV, HBV replication was only seen in the livers of mice that were infected with wild-type HBV (Tsuge et al, 2010; Tsuge et al, 2005). HBx expression also stimulates HBV replication in HepG2 cells, a human hepatoblastoma cell line, and in cultured primary rat hepatocytes [reviewed in (Bouchard & Schneider, 2004)]. Although most studies indicate that HBV replication in Huh7 cells, a human hepatoma cell line, is not regulated by HBx expression, a recent study demonstrated that HBx can also stimulate HBV replication in Huh7 cells (Lim et al, 2010). Many of the signaling pathways that are modulated by HBx, and will be discussed in the following sections of this chapter, have also been shown to influence HBV replication. HBx modulation of intracellular calcium signaling, activation of the Proline-rich tyrosine kinase 2 (Pyk2)/Focal adhesion kinase (FAK)-Src-Ras-Raf-MAPK signaling pathway, regulation of transcription pathways, interaction with ultraviolet DNA damage binding protein 1 (UVDDB1), and association with proteasome factors affect HBx regulation of HBV replication (Bouchard et al, 2001b; Gearhart & Bouchard, 2010b; Klein et al, 1999; Leupin et al, 2005; Tan et al, 2009; Tang et al, 2005; Zhang et al, 2004). Overall, studies in various model systems suggest that the X proteins of mammalian hepadnaviruses have an important stimulatory role during replication of mammalian hepadnaviruses.

#### **3.2 HBx and hepatocellular carcinoma**

108 Hepatocellular Carcinoma – Basic Research

was overexpressed out of the context of HBV replication, suggesting that studies in various cellular systems can provide valuable insights into HBx activities [reviewed in (Benhenda et al, 2009; Bouchard & Schneider, 2004; Koike, 2009)]. Moreover, because HBx is often expressed in HBV-associated liver tumor cells even when expression of other HBV proteins is not detectable (Wang et al, 2004b; Wollersheim et al, 1988), studies in immortalized or transformed cells may help identify HBx activities that could be present in transformed cells but absent in normal hepatocytes. Overall, it is likely that studies in immortalized or transformed cells, in cultured primary hepatocytes, in the livers of available animal models, and when HBx is expressed in the absence of other HBV proteins and in the context of HBV replication each contribute important information regarding the various activities of this protein. Considering the compact nature of the HBV genome, the limited number of proteins encoded by the HBV genome, and the necessity for these proteins to perform their functions in an environment that may not favor viral replication, it is not surprising that HBV proteins such as HBx, which is the only regulatory protein encoded by the HBV genome, have multiple functions that impact HBV replication and cellular physiology.

Many studies have indicated that replication of mammalian hepadnaviruses is stimulated by their respective X proteins. Two different studies performed in woodchucks showed that the woodchuck hepatitis virus (WHV) X protein (WHx) is absolutely required for WHV replication in the livers of woodchucks (Chen et al, 1993; Zoulim et al, 1994); however, in one dissenting study, another group detected a very low level of WHV replication in woodchucks infected with an WHx-deficient WHV mutant (Zhang et al, 2001). Evidence that HBx stimulates HBV replication has been generated from studies in various mouse models and cell culture systems. Transgenic mice were generated with either a wild-type HBV genome or a mutant form of the HBV genome that did not express HBx; HBV replication was detected in both the wildtype HBV and mutant HBV-transgenic mice (Xu et al, 2002). However, when the HBx-deficient HBV-transgenic mice were bred with HBxtransgenic mice, higher levels of circulating HBV viremia and higher levels of HBV core and envelope proteins were detected in the double transgenic mice than in their HBx-deficient, HBV-transgenic counterparts (Xu et al, 2002). These studies suggest that although HBx may not be absolutely required for HBV replication in this model system, HBx does enhance HBV replication in the livers of these mice. An important caveat to the studies in HBVtransgenic mice is that these mice do not produce nuclear HBV cccDNA and may not recapitulate all aspects of authentic HBV replication (Guidotti et al, 1995). Additional evidence that HBx stimulates HBV replication has come from a study where mice were hydrodynamically injected with a plasmid encoding the wild-type HBV genome or a mutant HBx-deficient HBV genome (Keasler et al, 2007). The results of these studies demonstrated that although HBx was not absolutely required for HBV replication, there was a very significant decrease in HBV replication in mice injected with the HBx-deficient HBV as compared to mice injected with the wildtype HBV genome (Keasler et al, 2007). Interestingly, when a plasmid encoding HBx was co-injected with a plasmid encoding the mutant HBx-deficient HBV genome, HBV replication levels were restored to those seen in mice injected with the plasmid encoding wild-type HBV (Keasler et al, 2007). Finally, studies in mice with humanized livers showed that after direct infection with wild-type HBV or an HBx-deficient HBV, HBV replication was only seen in the livers of mice that were infected

**3.1 HBx and HBV replication** 

HBx expression and activities are thought to be major contributing factors in the development of HBV-associated HCC; however, the exact contribution of HBx to HCC development is unknown. One important clue that implicates HBx in the development of HBV-associated HCC is that chronic infections of birds with avian hepadnaviruses are not associated with the development of HCC [reviewed in (Seeger et al, 2007)]. The avian hepadnaviruses either do not encode an X protein or encode an X protein that is highly divergent from the X proteins of mammalian hepadnaviruses (Mandart et al, 1984; Sprengel et al, 1988). The mammalian hepadnaviruses all encode an X protein, and only infections with mammalian hepadnaviruses are associated with the development of HCC in their respective hosts [reviewed in (Seeger et al, 2007)]. Additional evidence for the involvement of HBx in HCC development has come from studies in HBx-transgenic mice. Some studies have shown that HBx expression can directly cause HCC in HBx-transgenic mice (Kim et al, 1991; Yu et al, 1999). In contrast, other researchers have found that HBx expression alone is not sufficient for the development of liver tumors in HBx-transgenic mice but can sensitize these mice to chemical- or oncogene-induced HCC (Madden et al, 2001; Slagle et al, 1996; Terradillos et al, 1997; Zhu et al, 2004). While the reason for the observed differences in tumor development in the various HBx-transgenic mice has not been completely explored and may be related to the genetic background of the mice and/or the level of HBx expression, studies in these HBx-transgenic mice strongly support the notion that HBx can have at least a co-factor role in the development of HCC. Importantly, a co-factor role for HBx in the development of HBV-associated HCC is more consistent with the biology of HCC development in chronically HBV-infected individuals; development of HCC in chronically HBV-infected individuals can take decades to arise [reviewed in (Seeger et al, 2007)]. The observation that HBV-associated HCC requires decades to arise suggests that HBV does not encode a strongly oncogenic protein but instead encodes proteins that can cause subtle changes to hepatocyte physiology that could sensitize hepatocytes to other oncogenic signals [reviewed in (Bouchard & Navas-Martin, 2011; Neuveut et al, 2010)]. The activities of HBx that could influence HCC development are discussed below.

Modulation of Cell Proliferation Pathways by the Hepatitis B

development of HBV-associated HCC.

**3.4.2 HBx and p53** 

Virus X Protein: A Potential Contributor to the Development of Hepatocellular Carcinoma 111

et al, 2002). Disruption of the WHx-UVDDB1 interaction inhibited WHV replication in woodchucks, indicating that WHx and UVDDB1 must associate in order for WHV replication to occur *in vivo* (Sitterlin et al, 2000). WHx was also shown to interact with UVDDB2. The interaction between WHx and UVDDB1 stabilized WHx by protecting it from proteasomal degradation, but the interaction between UVDDB1 and UVDDB2 and between HBx and UVDDB2 counteracted this protection (Bergametti et al, 2002). In one study, HBx altered UVDDB1 activity, resulting in decreased cell viability (Lin-Marq et al, 2001). Similar to the required interaction of WHx and UVDDB1 for WHV replication, HBx association with UVDDB1 was also shown to be required for HBV replication (Sitterlin et al, 2000). HBx expression diminished the innate ability of cells to repair DNA damage, and the results of a study in HepG2 cells suggested that the binding of HBx to UVDDB1 inhibits normal UVDDB1 functions and can result in chromosome segregation defects (Becker et al, 1998; Martin-Lluesma et al, 2008). Results from studies in HBx-transgenic mice, however, indicated that the expression of HBx did not significantly increase the accumulation of spontaneous mutations, implying that although the interaction between HBx and UVDDB1 may inhibit the ability of cells to repair DNA damage, this interaction is not directly mutagenic (Madden et al, 2000). Interestingly, it has been suggested that the interaction between HBx and UVDDB1 may be involved in the HBx-related cell cycle arrest at the G1/S phase border [reviewed in (Bouchard & Schneider, 2004)]. Overall, a decreased ability to repair DNA damage caused by the interaction of HBx with the UVDDB complex, combined with modulation of cell proliferation pathways, could be important factors in the

The results of various studies suggest that HBx may interact with the tumor suppressor p53 both *in vivo* and *in vitro*. HBx that was tagged with glutathione S-transferase (GST-HBx) was shown to interact with *in vitro* translated p53; a reciprocal interaction between GST-p53 and *in vitro* translated HBx was also observed (Wang et al, 1994). p53 and HBx produced by *in vitro* translation and subsequently co-immunoprecipitated with either anti-HBx or anti-p53 antibodies also confirmed an interaction between p53 and HBx (Feitelson et al, 1993). Several reports indicate that HBx can alter the sequence-specific DNA binding and transcriptional regulatory capacity of p53 as well as p53 stimulation of apoptotic pathways (Elmore et al, 1997; Lee & Rho, 2000; Wang et al, 1994; Wang et al, 1995). The results of one study suggested that HBx can interact with the carboxy-terminal domain of p53 and that this interaction leads to inhibition of p53-induced apoptosis in normal primary human fibroblasts (Wang et al, 1995). Results of another study suggested that p53 can be sequestered in the cytoplasm by HBx, thus leading to the inhibition of p53-mediated apoptosis (Elmore et al, 1997). Interestingly, there are varying results regarding the role of p53 in the regulation of apoptosis by HBx; both p53-dependent (Wang et al, 2008) and p53 independent regulation of apoptosis by HBx have been observed (Shintani et al, 1999; Terradillos et al, 1998). Studies have also interrogated the influence of p53 on HBx regulation of the cyclin-dependent kinase inhibitor, p21; HBx increased the level of p21 in NIH3T3 cells in the presence of p53 but did not increase the level of p21 when p53 was knocked down (Ahn et al, 2002). However, in a different study, HBx increased p21 levels in Hep3B cells, a p53 mutant HCC cell line (Park et al, 2000). Since activities of HBx that were linked to p53-dependent pathways were also observed in the absence of p53, the functional

#### **3.3 Intracellular localization of HBx**

The primary intracellular location of HBx is the cytoplasm, although a small fraction of HBx can be found in the nucleus of cells (Doria et al, 1995; Haviv et al, 1998b; Henkler et al, 2001; Siddiqui et al, 1987; Urban et al, 1997; Vitvitski-Trepo et al, 1990). The localization of HBx is dependent on its level of expression. When HBx is expressed at very low levels, it is mainly localized to the nucleus, and when HBx is expressed at high levels, its localization is mainly cytoplasmic (Henkler et al, 2001). Many studies have also shown that a fraction of cytosolic HBx localizes to the mitochondria and can interact with the voltage-dependent anion channel (VDAC) 3, a component of the mitochondrial permeability transition pore (MPTP) (Clippinger & Bouchard, 2008; Henkler et al, 2001; Huh & Siddiqui, 2002; Kim et al, 2007; Rahmani et al, 2000; Shirakata & Koike, 2003; Takada et al, 1999). Cytoplasmic, nuclear, and mitochondrial HBx localization have been directly linked to HBx activities that regulate specific cellular signal transduction or transcription pathways [reviewed in (Ma et al, 2011)].

#### **3.4 HBx interacts with multiple cellular proteins**

HBx has been reported to interact with various cellular proteins, such as components of the proteasome complex (Fischer et al, 1995; Huang et al, 1996), UVDDB1 and 2 (Becker et al, 1998; Lee et al, 1995; Sitterlin et al, 2000)), the cell cycle regulatory protein p53 (Elmore et al, 1997; Feitelson et al, 1993; Wang et al, 1994), the NF-B inhibitory protein IB- (Weil et al, 1999), and VDAC3 (Rahmani et al, 2000; Rahmani et al, 1998), to name a few. HBx can also interact with components of the general transcription machinery such as the TATA-binding protein (TBP), the RBP5 subunit of RNA polymerase, and the general transcription factors TFIIB and TFIIH [(Aufiero & Schneider, 1990; Barnabas et al, 1997; Cheong et al, 1995; Haviv et al, 1998a; Haviv et al, 1998b; Maguire et al, 1991; Williams & Andrisani, 1995; Yang et al, 1999); reviewed in (Bouchard & Schneider, 2004)]. While the potential association of HBx with many cellular proteins could explain the multifunctional nature of HBx, because many studies that have analyzed HBx-interacting proteins were conducted in systems in which both HBx and these proteins were overexpressed, whether all of these interactions exist in normal hepatocytes when HBx is expressed during HBV replication remains to be determined. Due to space limitations, the following sections will focus on the interaction of HBx with UVDDB1 and 2 and p53; these interactions have been studied by various groups and may play an important role in HBV-induced HCC (Feitelson et al, 1993; Lee et al, 1995; Sitterlin et al, 2000; Wang et al, 1994). A more extensive discussion of proteins that may interact with HBx can be found in recently published reviews of this topic (Arbuthnot et al, 2000; Benhenda et al, 2009; Wei et al, 2010; Zhang et al, 2006).

#### **3.4.1 HBx and UVDDB1 and 2**

UVDDB1 associates with UVDDB2 to form the UV-DDB complex, which functions in nucleotide excision repair. The UVDDB complex also interacts with the transcription factor E2F1, indicating a role for the UVDDB complex in both DNA repair and cell cycle control [(Datta et al, 2001; Hayes et al, 1998); reviewed in (Butel et al, 1996)]. Results from yeast-2 hybrid screens indicated that HBx can directly interact with UVDDB1 (Lee et al, 1995; Lin-Marq et al, 2001; Sitterlin et al, 2000). Further evidence for an interaction between hepadnavirus X proteins and the UVDDB complex was provided from studies performed with WHV; these studies confirmed an interaction between WHx and UVDDB1 (Bergametti

et al, 2002). Disruption of the WHx-UVDDB1 interaction inhibited WHV replication in woodchucks, indicating that WHx and UVDDB1 must associate in order for WHV replication to occur *in vivo* (Sitterlin et al, 2000). WHx was also shown to interact with UVDDB2. The interaction between WHx and UVDDB1 stabilized WHx by protecting it from proteasomal degradation, but the interaction between UVDDB1 and UVDDB2 and between HBx and UVDDB2 counteracted this protection (Bergametti et al, 2002). In one study, HBx altered UVDDB1 activity, resulting in decreased cell viability (Lin-Marq et al, 2001). Similar to the required interaction of WHx and UVDDB1 for WHV replication, HBx association with UVDDB1 was also shown to be required for HBV replication (Sitterlin et al, 2000). HBx expression diminished the innate ability of cells to repair DNA damage, and the results of a study in HepG2 cells suggested that the binding of HBx to UVDDB1 inhibits normal UVDDB1 functions and can result in chromosome segregation defects (Becker et al, 1998; Martin-Lluesma et al, 2008). Results from studies in HBx-transgenic mice, however, indicated that the expression of HBx did not significantly increase the accumulation of spontaneous mutations, implying that although the interaction between HBx and UVDDB1 may inhibit the ability of cells to repair DNA damage, this interaction is not directly mutagenic (Madden et al, 2000). Interestingly, it has been suggested that the interaction between HBx and UVDDB1 may be involved in the HBx-related cell cycle arrest at the G1/S phase border [reviewed in (Bouchard & Schneider, 2004)]. Overall, a decreased ability to repair DNA damage caused by the interaction of HBx with the UVDDB complex, combined with modulation of cell proliferation pathways, could be important factors in the development of HBV-associated HCC.
