**5.1. Mechanisms regulating HBV replication**

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].

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).

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

18 Liver Cancer

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], resulting in epigenetic changes of virus gene expression. HBx binding to CUL4-DDB1 triggers altered enzymatic activity of the E3 ligase CRL4, which then stimulates the ubiquitination and subsequent proteasomal degradation of Smc5/6 [68–70], thereby promoting virus replication. Other anti-viral systems, such as IFN induced APOBEC3A [50], may also be similarly degraded. In this context, HBV is not very good in triggering innate immunity, which may underscore why there are hundreds of millions of carriers worldwide [71]. As mentioned above, sequestration and reverse transcription of pregenomic HBV RNA in immature nucleocapsids (**Figure 2**) may block the induction of innate immunity. In addition, although HBV replication is exquisitely sensitive to inhibition by IFNs, HBx appears to block IFN expression and signaling [72–74], suggesting that both innate and adaptive immunity could be compromised, thereby permitting virus persistence. Under these circumstances, CLD would continue to damage the liver while being unable to resolve the virus infection. HBx also regulates HBV replication by stimulating the expression of DNA methyl-transferases (DNMTs), which suppresses HBV transcription via DNA methylation [75]. DNMTs also methylate tumor suppressor genes, thereby down-regulating their expression, and permitting the accumulation of mutations and chromosomal instability that contribute importantly to HCC. Thus, HBx regulates the activity of ccc DNA in both positive and negative ways, and in doing so, impacts upon the pathogenesis of CLD. The reason why it is important to regulate the intrahepatic levels of ccc DNA is because when virus antigens are greatly overproduced, they could trigger cytopathic effects (CPE), thereby limiting virus replication. For example, mutations in the preS region of the S gene prevent secretion of surface antigen and complete virus particles, and eventually CPE. Pre-S mutations also promote recycling of viral DNA into the nucleus where it results in increased levels of viral ccc DNA, which potentially promotes virus persistence [76] (**Figure 2**). In transgenic mice overproducing HBsAg, CPE develops and eventually evolves into HCC [77]. Although the latter is not characteristic of HCC pathogenesis among human carriers, it does underscore that selected HBV mutants that may arise during chronic infection potentially contribute to pathogenesis via CPE.

dependent manner [84]. This is not an exhaustive list. Many of these molecules are turned on as a result of HBx stimulating multiple signal transduction pathways in the cytoplasm (in addition to NF-ĸB), but the bigger question is trying to understand how a non-cytopathic

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The fact that HBx plays a central role in HBV replication suggests that intracellular conditions that stimulate HBx activity would also promote the carrier state, which would be evolutionally selected for because it would provide a large window of time for virus to be transmitted to other hosts. In this context, the expression and activity of HBx is stimulated in an oxidative environment, since the addition of anti-oxidants to cells expressing HBx strongly diminish HBx *trans*-activation activity [85, 86]. An oxidative environment (accompanied by oxidative stress of cellular organelles) could be created in the infected cell by virtue of the association of HBx with mitochondria [87]. HBx interacts with the voltage dependent anion channel on the outer mitochondrial membrane, altering transmembrane potential [88], resulting in diminished electron transport, increased free radical accumulation, including elevated lipid peroxidation products [89], release of calcium into the cytosol [55], and under specific circumstances, cell death [90]. Release of calcium into the cytosol, resulted in the activation of the protein tyrosine kinase 2 and Src kinase families, leading to stimulation of ras, raf, mitogen activated protein kinase, and Jun, which stimulate HBV transcription and replication [55]. HBx also induces oxidative stress in the endoplasmic reticulum, which activates the unfolded protein response and expression of pro-inflammatory cyclooxygenase-2 through the activating transcription factor 4 pathway [91]. Free radicals are also characteristic of immune responses aimed at damaging and destroying infected cells that are replicating HBV. In addition, mitochondrial associated HBx induces oxidative stress, which activates selected transcription factors, such as NF-ĸB, STAT3 and activating protein 1 [86]. However, HBx is also known to block mitochondrial triggered cell death, not only by activation of survival [21, 92] and hepato-protective pathways such as NF-ĸB that over-ride apoptosis signaling, but also by blocking key caspases and promoting autophagy [93] and mitophagy [94]. The maintenance of mitochondrial and cellular homeostasis by mitophagy acts to attenuate virus induced apoptosis, so that on the one hand, autophagy and mitophagy promote cell survival and virus

persistence, while simultaneous mitochondrial damage may contribute to CLD [94].

In this chronic pro-inflammatory environment, one would expect to see a correlation between HBx staining and the intensity of CLD. In fact, WHx staining has been observed around inflammatory foci in chronically infected woodchuck livers [95], and among human carriers, relatively low levels of intrahepatic HBx staining was observed in patient biopsy samples from people with low grade hepatitis, while intense and widespread HBx staining was observed in patient biopsies from those with advanced fibrosis and cirrhosis [45, 96], suggesting a direct correlation between HBx staining and liver damage. Independent work also showed low levels of HBx mRNA in the livers of patients with mild CLD (e.g., mild hepatitis), and much higher levels among patients with severe lesions in the liver (advanced fibrosis and cirrhosis) [97]. The relationship of HBx expression to disease severity is also consistent with the idea that when the liver regenerates following each bout of hepatitis, fragments of HBV

**5.3. HBx and inflammation**

virus is mediating these and other related changes in infected cells.

#### **5.2. Oxidative damage and inflammation**

Although HBV is not cytopathic, HBx strongly activates NF-ĸB [78], which promotes the expression of many pro-inflammatory cytokines and chemokines that attenuate virus replication and contribute to the pathogenesis of CLD and HCC. For example, HBx stimulates the expression of IFN inducible proteins, such as the CXC chemokine IP-10 [79] which promotes leukocyte chemotaxis. HBx also stimulates production of interleukin-23 (IL-23) [79], which contributes to the maintenance and expansion of pro-inflammatory Th17 cells. Among others, IL-6 is up-regulated by HBx in a MyD88 manner [80], which indicates that HBx is activating a pro-inflammatory environment via innate immune pathways early on after infection. The repressive effect of IL-6 upon HBV replication is demonstrated by the fact that IL-6 treatment of infected cells results in the loss of HNF1a and HNF4a, both of which bind to ccc DNA. Il-6 also redistributes signal transducers and activators of transcription 3 (STAT3) signaling from ccc DNA to IL-6 target genes [49]. HBx targets up-regulation of IL-18, which up-regulates FasL [81], which in this case blocks the killing of infected cells by CTLs. HBx also up-regulates tumor TNFα [82], which was shown to suppress HBcAg expression [83], thereby inhibiting virus replication. In addition, the pro-inflammatory IL-32 was up-regulated by HBx in a NF-ĸB dependent manner [84]. This is not an exhaustive list. Many of these molecules are turned on as a result of HBx stimulating multiple signal transduction pathways in the cytoplasm (in addition to NF-ĸB), but the bigger question is trying to understand how a non-cytopathic virus is mediating these and other related changes in infected cells.

The fact that HBx plays a central role in HBV replication suggests that intracellular conditions that stimulate HBx activity would also promote the carrier state, which would be evolutionally selected for because it would provide a large window of time for virus to be transmitted to other hosts. In this context, the expression and activity of HBx is stimulated in an oxidative environment, since the addition of anti-oxidants to cells expressing HBx strongly diminish HBx *trans*-activation activity [85, 86]. An oxidative environment (accompanied by oxidative stress of cellular organelles) could be created in the infected cell by virtue of the association of HBx with mitochondria [87]. HBx interacts with the voltage dependent anion channel on the outer mitochondrial membrane, altering transmembrane potential [88], resulting in diminished electron transport, increased free radical accumulation, including elevated lipid peroxidation products [89], release of calcium into the cytosol [55], and under specific circumstances, cell death [90]. Release of calcium into the cytosol, resulted in the activation of the protein tyrosine kinase 2 and Src kinase families, leading to stimulation of ras, raf, mitogen activated protein kinase, and Jun, which stimulate HBV transcription and replication [55]. HBx also induces oxidative stress in the endoplasmic reticulum, which activates the unfolded protein response and expression of pro-inflammatory cyclooxygenase-2 through the activating transcription factor 4 pathway [91]. Free radicals are also characteristic of immune responses aimed at damaging and destroying infected cells that are replicating HBV. In addition, mitochondrial associated HBx induces oxidative stress, which activates selected transcription factors, such as NF-ĸB, STAT3 and activating protein 1 [86]. However, HBx is also known to block mitochondrial triggered cell death, not only by activation of survival [21, 92] and hepato-protective pathways such as NF-ĸB that over-ride apoptosis signaling, but also by blocking key caspases and promoting autophagy [93] and mitophagy [94]. The maintenance of mitochondrial and cellular homeostasis by mitophagy acts to attenuate virus induced apoptosis, so that on the one hand, autophagy and mitophagy promote cell survival and virus persistence, while simultaneous mitochondrial damage may contribute to CLD [94].

### **5.3. HBx and inflammation**

resulting in epigenetic changes of virus gene expression. HBx binding to CUL4-DDB1 triggers altered enzymatic activity of the E3 ligase CRL4, which then stimulates the ubiquitination and subsequent proteasomal degradation of Smc5/6 [68–70], thereby promoting virus replication. Other anti-viral systems, such as IFN induced APOBEC3A [50], may also be similarly degraded. In this context, HBV is not very good in triggering innate immunity, which may underscore why there are hundreds of millions of carriers worldwide [71]. As mentioned above, sequestration and reverse transcription of pregenomic HBV RNA in immature nucleocapsids (**Figure 2**) may block the induction of innate immunity. In addition, although HBV replication is exquisitely sensitive to inhibition by IFNs, HBx appears to block IFN expression and signaling [72–74], suggesting that both innate and adaptive immunity could be compromised, thereby permitting virus persistence. Under these circumstances, CLD would continue to damage the liver while being unable to resolve the virus infection. HBx also regulates HBV replication by stimulating the expression of DNA methyl-transferases (DNMTs), which suppresses HBV transcription via DNA methylation [75]. DNMTs also methylate tumor suppressor genes, thereby down-regulating their expression, and permitting the accumulation of mutations and chromosomal instability that contribute importantly to HCC. Thus, HBx regulates the activity of ccc DNA in both positive and negative ways, and in doing so, impacts upon the pathogenesis of CLD. The reason why it is important to regulate the intrahepatic levels of ccc DNA is because when virus antigens are greatly overproduced, they could trigger cytopathic effects (CPE), thereby limiting virus replication. For example, mutations in the preS region of the S gene prevent secretion of surface antigen and complete virus particles, and eventually CPE. Pre-S mutations also promote recycling of viral DNA into the nucleus where it results in increased levels of viral ccc DNA, which potentially promotes virus persistence [76] (**Figure 2**). In transgenic mice overproducing HBsAg, CPE develops and eventually evolves into HCC [77]. Although the latter is not characteristic of HCC pathogenesis among human carriers, it does underscore that selected HBV mutants that may

arise during chronic infection potentially contribute to pathogenesis via CPE.

Although HBV is not cytopathic, HBx strongly activates NF-ĸB [78], which promotes the expression of many pro-inflammatory cytokines and chemokines that attenuate virus replication and contribute to the pathogenesis of CLD and HCC. For example, HBx stimulates the expression of IFN inducible proteins, such as the CXC chemokine IP-10 [79] which promotes leukocyte chemotaxis. HBx also stimulates production of interleukin-23 (IL-23) [79], which contributes to the maintenance and expansion of pro-inflammatory Th17 cells. Among others, IL-6 is up-regulated by HBx in a MyD88 manner [80], which indicates that HBx is activating a pro-inflammatory environment via innate immune pathways early on after infection. The repressive effect of IL-6 upon HBV replication is demonstrated by the fact that IL-6 treatment of infected cells results in the loss of HNF1a and HNF4a, both of which bind to ccc DNA. Il-6 also redistributes signal transducers and activators of transcription 3 (STAT3) signaling from ccc DNA to IL-6 target genes [49]. HBx targets up-regulation of IL-18, which up-regulates FasL [81], which in this case blocks the killing of infected cells by CTLs. HBx also up-regulates tumor TNFα [82], which was shown to suppress HBcAg expression [83], thereby inhibiting virus replication. In addition, the pro-inflammatory IL-32 was up-regulated by HBx in a NF-ĸB

**5.2. Oxidative damage and inflammation**

20 Liver Cancer

In this chronic pro-inflammatory environment, one would expect to see a correlation between HBx staining and the intensity of CLD. In fact, WHx staining has been observed around inflammatory foci in chronically infected woodchuck livers [95], and among human carriers, relatively low levels of intrahepatic HBx staining was observed in patient biopsy samples from people with low grade hepatitis, while intense and widespread HBx staining was observed in patient biopsies from those with advanced fibrosis and cirrhosis [45, 96], suggesting a direct correlation between HBx staining and liver damage. Independent work also showed low levels of HBx mRNA in the livers of patients with mild CLD (e.g., mild hepatitis), and much higher levels among patients with severe lesions in the liver (advanced fibrosis and cirrhosis) [97]. The relationship of HBx expression to disease severity is also consistent with the idea that when the liver regenerates following each bout of hepatitis, fragments of HBV 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 contrast, the copy number of ccc DNA per cell decreases with regeneration.

**6. Conclusions**

and contribute centrally to malignant transformation.

**Acknowledgements**

to work on this project.

**Conflict of interest**

The author declares no conflict of interest.

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,

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The author wants to acknowledge Temple University for providing him with the opportunity

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