**4. Relationship between persistent virus replication, integration of HBV DNA, and the risk for the development of HCC**

There is evidence to suggest that persistent, high levels of HBV replication correlate with the progression of CLD to HCC [34]. However, independent work showed an elevated risk for HCC among patients with CLD but low virus titers [35, 36]. Other observations have shown no correlation between HBV DNA levels in serum (>10<sup>5</sup> copies/ml) and histological grade or stage of liver disease in carriers [37, 38]. In addition, it is controversial as to whether long term nucleoside analog therapy resulted in a decreased risk for the development of HCC [6, 35]. Given that HBV is not directly cytopathic [39], that carriers with high levels of HBV DNA in serum are often asymptomatic, and that the pathogenesis of CLD is immune mediated [17, 40], a correlation between virus replication and CLD may contribute to, but not by itself, determine disease progression. Moreover, most carriers with CLD who develop cirrhosis and HCC have long since seroconverted from HBeAg (reflecting high levels of virus replication) to anti-HBe (reflecting low or undetectable virus replication), indicating that disease progression may occur at low virus titers [36]. Among patients with sustained high levels of HBV replication and successive bouts of CLD, there is a wave of liver regeneration following each episode of hepatitis to restore full liver function. At these times, fragments of HBV DNA, mostly encoding the HBx ORF (and sometimes the HBx plus preS/S ORFs), become integrated at multiple sites within host DNA [41, 42] (**Figure 3**). Over time, these integration events result in increased intrahepatic expression levels of HBx that alter patterns of host (and support virus) gene expression (**Figure 3**). HBV integrates early after infection, not only in permissive liver cell lines, but also in non-replicating primary human hepatocytes [43]. Many fragments of integrated HBV DNA encode HBx that is capable of *trans*-activation [44]. Although the relatively low levels of HBx made from the virus mini-chromosome support virus gene expression and replication, it is hypothesized that as intrahepatic levels of HBx increase [45] (**Figure 3**), it epigenetically alter the expression patterns of selected host genes [46] that contribute to both virus persistence and to malignant transformation. Thus, the changing intrahepatic levels of HBx promote virus persistence and ultimately, contribute to malignant transformation [47].

nuclear deaminase resulted in a modest reduction in ccc DNA copy number via deamination [50]. Gene editing approaches, such as CRISPR/Cas9 have also been demonstrated to work *in vitro* and *in vivo* [51], but off-target effects, ability to access and act on all susceptible cells, and recognition of all HBV genotypes, remain to be addressed. In addition, the recent finding of ccc host DNA in both normal and tumor cells, as a mechanism whereby host cells regulate gene expression [52], implies that targeting ccc DNA may also have toxic effects on the treated cells whether or not they are virally infected. Thus, it is not clear whether this approach in a liver which is already damaged will exacerbate that damage and/or have an anti-tumor effect.

Pathogenesis of Hepatitis B Virus Associated Chronic Liver Disease

http://dx.doi.org/10.5772/intechopen.79746

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HBx, the *trans*-activation protein of HBV, *trans*-activates virus gene expression and replication *in vitro* [54, 55]. The contribution of this regulatory protein to virus persistence in the carrier state was shown in woodchucks experimentally infected with the HBV-like virus, woodchuck hepatitis virus (WHV). Wild type WHV readily establishes a chronic infection, characterized by persistent virus replication and CLD that progresses to HCC [56]. However, experimental infection with a mutant of WHV that does not encode woodchuck hepatitis x (WHx) antigen yielded no carrier state and no CLD [57, 58], suggesting that *trans*-activation of virus gene expression and replication is central to the establishment of the carrier state. Among infected woodchucks, there was co-staining between WHV core antigen (where virus replication takes place) and WHx [59, 60], while in human infection, HBx often co-existed with HBe in serum [61] and replication complexes (i.e., with HBcAg) in the liver [62]. Thus, HBx expression is

At the molecular level, HBx regulates HBV replication by binding to various cellular proteins. For example, HBx binds to jumonji C-domain-containing 5 (JMJD5), a arginyl-hydroxylase, which promotes the expression of transcription factors (e.g., such as hepatocyte nuclear factors 3 gamma and 4 alpha [HNF3G and HNF4A] and CCAAT/enhancer-binding protein alpha) that facilitate hepatocyte differentiation [63]. Given that HBV replicates in differentiated hepatocytes, the binding of HBx to JMJD5 facilitates HBV replication via epigenetic alterations in host gene expression. In addition, HBx promotes the formation of ccc DNA by recruiting the transcriptional scaffold, p300; the cAMP response element binding protein CREB; the CREB transcription factor binding protein, CBP; the histone acetyltransferase p300/CBP-associated factor, as well as the histone deactylases HDAC1, Sirt1 [48] and Sirt2 [12]. Once ccc DNA is formed, HBx up-regulates HBV replication, in part, by binding to cullin4-damage specific DNA binding protein (CUL4- DDB1) ubiquitin ligase [64, 65], suggesting that HBx may function, at least in part, at the level of the proteasome. HBx modulates proteasome activity by direct binding to the 26S proteasomal subunit [66], which is responsible for degradation of HBx and several anti-viral proteins. One of the latter is Smc5/6, which is involved in the structural maintenance of chromosomes (i.e., genome stability) and DNA repair [67]. Smc5/6 and HBx bind to the HBV mini-chromosome [67, 68],

**5. Contribution of HBx to pathogenesis of CLD by regulation of** 

**HBV replication**

associated with virus replication.

**5.1. Mechanisms regulating HBV replication**

#### **4.1. Covalently closed circular HBV (ccc) DNA**

Given that the current treatment of chronic hepatitis B with nucleoside analogs is not curative, there has been a major effort to eliminate ccc DNA [47], especially since ccc DNA is the template for all virus transcripts. Since nucleoside analogs do not eliminate integrated HBV templates or the HBV mini-chromosome, continued virus gene expression from these templates will drive pathogenesis toward HCC. Formation of ccc DNA is a complex process that involves a variety of host proteins, including several DNA polymerases [48] that could potentially be therapeutic targets, although this approach may be accompanied by toxicity. As outlined below, HBx regulates the formation, function and intracellular copy number of ccc DNA by several epigenetic mechanisms that involve altered expression of histone methyltransferases and histone deacetylases, by promoting degradation of the anti-viral restriction factor Smc5/6, and by increasing expression of DNA methyltransferases [48]. Anti-viral immune responses in which selected cytokines mediate non-cytolytic degradation of ccc DNA have also been documented *in vitro* [48, 49]. Among these, IFN alpha up-regulated expression of APOBEC3

**Figure 3.** Natural history of chronic hepatitis B featuring the progressive lesions that develop in CLD compared to increased number of integration events, many of which produce functional HBx (modified from [53] with permission).

nuclear deaminase resulted in a modest reduction in ccc DNA copy number via deamination [50]. Gene editing approaches, such as CRISPR/Cas9 have also been demonstrated to work *in vitro* and *in vivo* [51], but off-target effects, ability to access and act on all susceptible cells, and recognition of all HBV genotypes, remain to be addressed. In addition, the recent finding of ccc host DNA in both normal and tumor cells, as a mechanism whereby host cells regulate gene expression [52], implies that targeting ccc DNA may also have toxic effects on the treated cells whether or not they are virally infected. Thus, it is not clear whether this approach in a liver which is already damaged will exacerbate that damage and/or have an anti-tumor effect.
