**6. Conclusions**

DNA encoding the HBx region (and sometimes part of the preS/S encoding gene as well) increasingly integrate into multiple regions of the host genome during normal host DNA replication, resulting in increasing accumulation of intrahepatic HBx as CLD progresses. In

The relationship between HBx expression and CLD has been recapitulated in HBx transgenic mice, where the presence, frequency and distribution of HBx in the liver increase with age, as does liver pathology, which progressively develops from hepatitis and steatosis, to dysplasia and microscopic nodules of HCC, and finally to multi-nodular macroscopic HCC with age [98]. In this model, HBx is expressed from its own enhancer and promoter, which is not active until after birth when appropriate transcription factors in the liver begin to appear. HBx expression triggers immune responses in the absence of other HBV gene products, so it is likely that the pathogenesis observed is due to the impact of increasing levels of HBx upon host gene expression combined with immune responses directed against virus infected cells. There is no ccc DNA in this system, just as it is difficult to detect HBV replication among patients with advanced stages of CLD (i.e., cirrhosis). Thus, it is possible that early in chronic infection, and immune responses to virus antigens emanating from ccc DNA templates play an important role in triggering and sustaining immune mediated pathogenesis, but following bouts of CLD and liver regeneration, where the levels of virus replication decrease at the same time that integration of virus DNA fragments increase, pathogenesis appears to be increasingly driven by one or more antigens made from integrated HBV DNA. Although *cis*-acting mechanisms have been postulated to contribute importantly to the pathogenesis of HCC in selected cases, the broadly distributed integration events of the HBx ORF into most chromosomes [99], suggests that the HBx proteins encoded by most integration events promote CLD and HCC in *trans* [47]. In this model, integration of HBV sequences would accumulate in areas of euchromatin and fragile sites much more frequently that at or within specific genes [100]. The model above suggests that targeting ccc DNA in HBeAg carriers with CLD may be an important therapeutic goal to bring about a functional (but not sterilizing) cure due to the presence of integrated virus DNA that express one or more virus proteins. Among anti-HBe carriers with advanced CLD, targeting the much lower levels of ccc DNA may not be effective in preventing progression to cirrhosis and HCC, because at this stage, most of the HBx made probably comes from integrated templates. Under these circumstances, ccc DNA may persist in a transcriptionally inactive form, which is consistent with the absence of HBV DNA in the blood, even after treatment with direct acting anti-viral agents or therapy aimed at stimulating immune responses against virus infected cells [101, 102]. In fact, early work already pointed out that seroconversion to anti-HBe is sometimes associated with the progression of CLD [103, 104], even though later work showed that disease progression was associated with continued replication of HBV DNA carrying one or more mutations in the core gene that blocks production of HBeAg [27]. These mutations were probably selected for during the natural history of infection by immune responses targeting HBcAg [105]. Although these findings suggest that CLD progresses in the liver supporting replication of selected virus mutants, it has also recently been suggested that linear HBV DNA, and not ccc DNA, is the template for integration into host DNA [43], from which one or more virus gene products are made, and contribute to pathogenesis. Thus, persistent inflammation in a chronically damaged liver may result in the

contrast, the copy number of ccc DNA per cell decreases with regeneration.

22 Liver Cancer

development of HCC despite low levels or undetectable levels of virus replication.

HBV encodes polypeptides from four ORFs that trigger corresponding immune responses during acute and chronic infections. When these responses are rapid, strong and multi-specific, acute, resolving infection can be achieved. When these immune responses are weak and of limited specificity (against few virus epitopes), the carrier state may develop. Although the pathogenesis of HBV is variable in different hosts, the virus encodes proteins that blunt innate immunity, and as a consequence, adaptive immunity is not triggered at all or to a limited extent. The latter causes liver damage over many years without eliminating the virus. Even though available treatments suppress virus replication, none are curative, and the persistence of viral ccc DNA sustains infection. Production of HBx regulates virus gene expression and replication, but over time, increased integration of HBV DNA fragments encoding HBx results in high levels of HBx expression that epigenetically alter the expression of numerous host genes that up- or down-regulate HBV replication and impact disease activity. For example, HBx activation of AKT decreased HBV replication, but this was accompanied by an inhibition of apoptosis, suggesting that HBx balances HBV replication and cell survival by stimulating signaling that enhance hepatocyte survival at the expense of higher levels of HBV replication [106]. The generation of free radicals by immune responses against virus infected cells, combined with HBx mediated alterations in mitochondrial function, promote HBx activity. These events result in the activation of signaling pathways (e.g., AP-1 and NF-ĸB) that over-ride apoptosis and/or directly block the activation of critical caspases, so that whether HBx stimulates or block apoptosis depends upon whether the liver is experiencing inflammation and oxidative stress. It also depends upon whether HBx is being expressed in normal hepatocytes, where apoptotic pathways could be triggered, or whether HBx is expressed at high levels in cells where apoptotic pathways are compromised. In addition to being pro-inflammatory, activated NF-ĸB protects infected cells against immune elimination. Thus, the dichotomy of HBx activity may be a reflection of the environment wherein HBx is expressed. Importantly, the epigenetic mechanisms whereby HBx regulates virus replication also have an impact on cell growth and survival, and many of these same alterations in host gene expression are also hallmarks of cancer [107], which may explain why there is such a high risk of HCC among carriers with CLD [11]. The common denominator is that many of the pathways and molecules that support HBV gene expression and replication also protect infected cells from elimination, and contribute centrally to malignant transformation.
