Inhibition of interferon signaling during Hepatitis B Virus infection and virus-associated hepatocellular carcinoma

Abstract

Chronic Hepatitis B Virus (HBV) infection is linked to hepatocellular carcinoma (HCC) pathogenesis. The World Health Organization estimates that globally 257 million people are chronic HBV carriers at risk of developing liver cancer. Current therapies for prevention and treatment of HCC are inadequate. Although interferon-based treatment strategies hold great promise for combating chronic infection and HCC, many patients do not respond to the IFN-based drugs for reasons not completely understood. Interferon signaling plays key roles in activation of innate and adaptive immunity. However, HBV has evolved various mechanisms to evade IFN signaling. In this review, we present the basics about HBV infection and interferon signaling. Next, we discuss mechanisms through which HBV downregulates the function -activity and transcription- of the transcription factor STAT1 during acute and chronic infection. STAT1 is activated in response to all types (I/II/III) of interferon signaling and is essential in mediating all types (I/II/III) of interferon responses. Lastly, we discuss emerging evidence from different human cancers linking loss of interferon signaling to aggressive cancer and cancer stem cells. Whether the same occurs during HBV-associated hepatocarcinogenesis is discussed and currently under investigation.

Hepatitis B Virus:

Hepatitis B virus is a noncytopathic, hepatotropic virus that belongs to the hepadnaviral family. Following acute infection of the liver, 90–95% of the patients clear the infection and fully recover. The remaining 5–10% of the patients develop chronic HBV infection, and are at greater risk of developing hepatocellular carcinoma (HCC)[1], which is usually fatal. The World Health Organization (WHO) estimates that 257 million people worldwide are chronically infected with HBV, with approximately 887,000 deaths every year due to HBV/HCC complications [2].

HBV is a partially double-stranded DNA virus that infects hepatocytes. It has a compact genome of 3.2 kb encoding four proteins: the Core antigen (HBc), the DNA Polymerase (P), Surface (S) and X (HBx) proteins [3]. The HBV receptor is the Sodium Taurocholate Co-transporting Polypeptide (NTCP) protein, a hepatocyte transmembrane protein. HBV binds the NTCP receptor and becomes internalized [4]. The viral capsid is then transported to the nucleus where the partially double-stranded DNA is repaired to form the covalently closed circular (ccc)DNA [5–7]. In HBV-associated liver tumors the viral DNA is integrated into the host genome following DNA repair. cccDNA assumes a minichromosome-like structure, serving as the template for viral transcription. The mRNA transcripts are exported to the cytoplasm and used for translation of the HBV proteins. The longest transcript, the pre-genomic RNA (pgRNA), also functions as the template for viral replication, which occurs within nucleocapsids in the cytoplasm [3]. Nucleocapsids are enveloped during their passage through the endoplasmic reticulum (ER) and/or Golgi complex and secreted from the cell (Fig. 1)

Figure 1:

Diagram highlights key steps of the HBV life cycle: viral entry and uncoating, nuclear transport of the viral genome, formation of cccDNA, viral transcription and replication, viral assembly and release.

Prevalence, Transmission, Risks and Prevention:

According to the WHO [2], HBV has highest prevalence in Western Pacific and African regions, followed by the Eastern Mediterranean, the South-East Asia and the European Regions (Fig. 2). HBV is a blood-borne pathogen, transmitted by blood and other bodily fluids. In highly endemic regions, perinatal transmission (spread from mother to child at birth) is the major route of transmission. Since HBV is capable of surviving and causing infection for at least 7 days outside the human body, transmission may also occur via percutaneous or mucosal exposure to infected blood and various bodily fluids [8–10]. The risk of developing chronic HBV infection after acute exposure ranges from 80–90% in newborns of HBeAg-positive mothers; 30–50% in children (under six years age) and less than 5% in adults [11–15]. In general, immunosuppressed persons have higher chance of developing chronic HBV infection [16]. The HBV vaccine, available since 1982, can prevent chronic infection in 95% of recipients and should be administered within 24 hrs of birth[17].

Figure 2.

Global HBV infection patterns.

Treatment:

Current treatment options for chronic HBV infection [18–20] are (i) immune modulator drugs, (ii) antiviral drugs, and (iii) liver transplantation. Immune modulator drugs are interferon (IFN)-type drugs that boost the immune system, while antiviral drugs include nucleoside analogs that halt HBV replication by inhibiting the viral polymerase. Currently [20], there are eight approved drugs for hepatitis B in the United States - two types of injectable interferons (Pegylated-IFN-α and IFN-α), and six oral antivirals (Tenofovir dipovoxil fumarate, Tenofovir alafenamide, Entecavir, Telbivudine, Adefovir and Lamivudine). Studies have found that IFN-α treatment reduced the incidence of cirrhosis, and liver-related deaths [21]. Unfortunately, a large percentage of chronic HBV patients do not respond to IFN treatment for reasons that remain to be understood [18]. Regarding the effect of nucleoside analogs, viral resistance limits their long term success [22–24]. Furthermore, persistance of cccDNA is mainly responsible for recurrence of HBV in patients, even years after anti-viral therapy [25]. Liver transplantation can overcome the pitfalls of immune modulator drugs and antiviral drugs, however, the procedure carries significant risks. Moreover, it is not an option for everyone, and there are millions of people chronically infected by HBV. Hence, we need to identify new strategies to effectively treat chronic HBV patients.

HBV infection and activation of the innate immune response:

HBV persistence or viral clearance is dependent on the host immune response. Immediately after infection most viruses activate the innate immune response mediated by IFNs, as an early defense mechanism to inhibit viral replication, and thus limit the spread of the infection. Regarding HBV infection and activation of the innate immune response, studies in experimentally infected chimpanzees, also a natural HBV host, demonstrated absence of activation of the innate immune response, namely, IFN production and induction of Interferon Stimulated Genes (ISGs)[26]. Similar studies in humans are not available, because HBV infection is usually detected 10–12 weeks after the patient becomes infected. The results derived from the experimental infection of chimpanzees were interpreted to mean that HBV is a stealth virus [26, 27]. Based on the lifecycle of HBV and because viral replication occurs within the nucleocapsid, it was proposed that the virus has evolved mechanisms to hide recognition of viral nucleic acids by the cellular innate response sensors [27]. However, one must also consider that viral proteins are in the cytoplasm of infected cells. Interestingly, in vitro studies employing non-trasformed/non-neoplastic human HepaRG cells, transduced with baculovirus carrying the full length HBV genome, demonstrated that a strong HBV replication does/can induce a robust and HBV-specific innate immune response [28]. These results challenge the idea that HBV is a stealth virus [29]. In addition, many studies have identified mechanisms by which viral proteins counteract the innate immune response, reviewed in [29], and also discussed herein, partially explaining the “stealthy “ nature of HBV [30].

New HBV infection cellular and animal models have been established which have allowed a more in depth understanding of the mechanisms of these virus/host interactions. An in depth description of these HBV infection models can be found in the recent and comprehensive review by Allweiss and Dandri [31]. Some of these in vitro and in vivo animal models have been used to investigate how HBV bypasses cellular antiviral mechanisms, with focus on IFN signaling, as will be discussed herein.

The IFN signaling pathways:

In response to viral infection, the host cell releases cytokines called “Interferons”. All classes of interferons (IFNs I/ II and III) signal through the JAK/STAT pathway which results in transcriptional activation of Interferon Stimulated Genes (ISGs)[32]. IFN receptors usually exist as heterodimers. The cytoplasmic domain of all IFN receptors is bound by inactive Janus kinases (JAKs). Following IFN binding, the IFN receptors undergo conformational change bringing the inactive JAKs in close proximity. This results in auto-phosphorylation and activation of JAKs. Activated JAKs phosphorylate specific tyrosine residues in specific STATs (STAT1–5) leading to their activation (Fig. 3). Activated STATs undergo homo/hetero-dimerization and translocate to the nucleus via the Importin-RanGDP complex [33, 34]. STATs activate transcription of ISGs by binding to consensus DNA motifs in the promoter region of ISGs. Nuclear phosphatases (SHP-2, TC45) dephosphorylate STATs leading to STAT inactivation, and subsequent transport out of the nucleus by the exportin-RanGTP complex [35, 36].

Figure 3:

Diagram outlines the key features of type I/II/III IFN signaling pathways. The blue boxes indicate STAT1, while the red circles indicate the activating phosphorylation of all STATs. STAT1 can be activated by all types of IFN signaling, and is essential for all types of IFN responses. STAT1 homodimers bind to the GAS sequence.

STAT1 is the only member of the STAT family of transcription factors that can be activated by all types of IFNs (I/II/III), and significantly, STAT1 is a required component in the signal transduction mechanism of all IFN (I/II/III) types (Fig. 3). Specifically, IFN-II binding to its receptor promotes homodimerization of phosphorylated STAT1 which translocates to the nucleus and binds to Gamma-IFN–activated sequence (GAS) sequence upstream of ISGs[37]. IFN types I and III signal via heterodimers of phosphorylated STAT1 and STAT2 in association with IRF9 (Fig. 3). These heterodimers translocate to nucleus and bind to IFN-stimulated response elements (ISRE) in the promoters of ISGs [38].

HBV infection and IFN signaling:

Initially, in vitro cell culture studies identified various mechanisms by which HBV could interfere with IFN signaling. For instance, the HBV core protein (HBc) inhibits transcription of Ifnβ [39, 40] and MxA upon IFN-α stimulation [41]; the C-terminal domain of the viral polymerase inhibits expression of MyD88 [42], and the HBV X and S proteins block nuclear import of phosphorylated STAT1 via upregulation of protein phosphatase 2A (PP2A) [43].

Employing animal models of HBV infection, various studies have examined HBV effects on IFN-signaling. HBV infected and uninfected human liver chimeric mice (uPA/SCID) were treated with human IFN-α [44]. Changes in viral DNA and expression of human ISGs were measured by RT-PCR, and immunohistochemistry. Although IFN-α activated expression of human ISGs in hepatocytes that repopulated the livers of uninfected mice, it failed to increase expression of ISGs in HBV-infected mice, and did not induce nuclear translocation of phospho-STAT1 in HBV-infected human hepatocytes. Thus, HBV prevents induction of IFN-α signaling genes by inhibiting nuclear import of activated STAT1, although in this study the level of total, non-activated/unphosphorylated STAT1 was not assessed. On the other hand, it was shown, using both HBV replicating HepG2 cells and human liver chimeric mice (uPA/SCID) infected with HBV, that IFN-α inhibits HBV replication by decreasing viral transcription from the cccDNA minichromosome. Administration of IFN-α resulted in hypoacetylation of histones bound to cccDNA, and recruitment of transcriptional corepressors HDAC1, SIRT1, EZH2, and YY1 to the cccDNA. IFN-α treatment reduced binding of STAT1 and STAT2 to cccDNA. Furthermore, an HBV mutant lacking the STAT1/STAT2 DNA binding could not be repressed by IFN-α. These results demonstrate how IFN-α antagonizes viral biosynthesis by epigenetically repressing STAT1/2-mediated transcription from the cccDNA [45].

STAT1 regulation and HBV infection:

Since STAT1 is required for all types (I/II/III) of interferon signaling pathways (Fig. 3), the inhibition of STAT1 activation by HBV [43–45], identifies STAT1 as a critical cellular inhibition target by HBV infection. However, the mechanism (s) and cellular contexts by which HBV interferes with STAT1 activity are not fully understood. It was shown that arginine methylation of STAT1 by arginine methyltransferase 1 (PRMT1) modulates transcription of ISGs [46], and that protein phosphatase 2A (PP2A) inhibits PRMT1 [47]. In turn, hypomethylated STAT1 is less active because it is bound by its inhibitor PIAS1 [48, 49]. Interestingly, the levels of PP2A were found elevated in biopsy tissue of chronic HBV patients [50], suggesting that PP2A induction has a primary role in HBV-mediated inhibition of IFN signaling. More recent studies have identified via an RNAi screen, the methyltransferase SETD2 as the enzyme that also methylates STAT1 on lysine-525 [51]. It was shown that this is a critical modification in promoting the IFN-activated STAT1 phosphorylation, and the antiviral IFN response. Conditional knockout mice with hepatocyte-specific deletion of Setd2 exhibited enhanced HBV infection. In addition, chromatin immunoprecipitation assays demonstrated that SETD2 mediates the trimethylation of H3K36 bound to ISG promoters, including ISG15. This study identified SETD2 as an essential player in the IFN antiviral response. However, how HBV infection inactivates and counteracts SETD2 to allow virus biosynthesis is currently unknown.

Another mechanism that regulates STAT1 function involves the nuclear E3 ligase RNF2 as an inhibitor of IFN function by promoting STAT1 dissociation from DNA [52]. Specifically, it was shown that RNF2 directly bound to STAT1 after IFN stimulation and increased K33-linked polyubiquitination of the DNA-binding domain of STAT1 at position K379. This post-translational modification of STAT1 promoted dissociation of STAT1/STAT2 from DNA, and suppressed transcription of ISGs [52]. Whether this mechanism of regulation is operational during HBV infection is unknown.

Other molecules involved in the antiviral/innate immune response include viral nucleic acid sensors such as RIG-I [53]. RIG-I can detect viral RNAs in the cytoplasm during infection with a variety of viruses, including HBV [54]. Studies by Sato et al [55] demonstrated that RIG-I senses the stem-loop secondary structure of 5’-ε region of the HBV pgRNA, and interferes with its interaction with the viral polymerase, thereby suppressing viral replication. Overexpression of RNA containing the 5’-ε region in the liver of chimeric mice with humanized liver inhibited HBV replication by binding to the viral polymerase and competing/blocking polymerase binding to pgRNA. Thus, these studies showed that RIG-I functions not only as an RNA sensor but also as a host antiviral factor for HBV infection. Interestingly, RIG-I, which is an ISG, is the most significantly downregulated ISG in HCC, including poor prognosis HBV-related HCC [56]. It was shown [56] that RIG-I facilitated the nuclear translocation of activated STAT1, by reducing the association of STAT1 with its negative regulator SHP-1 [56]. The significance of reducing nuclear STAT1 in hepatocarcinogenesis is not understood. Moreover, whether this mechanism exerts any effect on STAT1 nuclear translocation during HBV infection is unknown.

Chronic HBV infection and the innate immune response:

Chronic HBV infection is linked to liver pathology, including fibrosis, cirrhosis and hepatocellular carcinoma. Although the mechanism responsible for establishment of chronic infection is not well understood, a likely cause is suppression of host immune responses. The examples discussed earlier demonstrate how HBV inhibits the innate response in the context of experimental HBV infection, modeling acute infection in patients. In this section studies will be presented on the effect of HBV infection on interferon signaling in chronically infected patients.

The Zoulim group [50] analyzed a large number of chronic HBV patients for intrahepatic expression of 67 genes that belong to multiple pathways of the innate immune response. The expression data of these ISGs were correlated to markers of viral infection in the serum and in liver biopsy tissue, including quantification of the S antigen (HBs), total HBV DNA, pgRNA, and cccDNA. The results demonstrate that chronic HBV infection suppresses immune responses of type I/II/III IFN signaling, including transcription of STAT1, in some patients [50]. Significantly, the suppression is more pronounced with higher levels of viral HBs antigen, which can be transcribed either from cccDNA or the integrated HBV DNA that occurs during hepatocellular carcinoma. In addition, several genes were found to be upregulated, including the PP2A phosphatase that was shown earlier to inhibit STAT1 signaling [43], the DNA methyltransferase DNMT3A that can silence viral and host genes including tumor suppressor genes and immunoregulatory genes [57], and STAT2, which was shown to suppress ISGs in hepatocytes after IFN stimulation [58]. Thus, these studies highlight the significance of chronic HBV infection in suppressing innate immunity, which influences both viral clearance and also pathways associated with anti-tumor immunity and apoptosis [59].

In terms of viral clearance, a major issue is the persistence of the HBV cccDNA in infected hepatocytes. Lucifora et al [60] presented studies of how IFN-α treatment of HBV infected primary hepatocytes induces specific degradation of cccDNA without hepatotoxicity, by upregulating the cytidine deaminases APOBEC3A and APOBEC3B. Cytidine deamination of cccDNA and formation of apurinic sites results in cccDNA degradation and clearance of HBV reactivation, without cytolysis. Repair of apurinic DNA involves the base excision repair (BER) mechanism which removes DNA lesions and maintains genomic integrity [61]. Interestingly, chronic HBV infected patients that respond to IFN treatment in comparison to non-responders, exhibit downregulated expression of the BER gene NEIL3; conversely, the APOBEC3A expression level was significantly increased [62]. This inverse correlation between APOBEC3A and NEIL3 expression during IFN treatment suggests that absence of cccDNA repair by the BER mechanism is important in cccDNA degradation. How IFN-α suppresses NEIL3 expression is unknown. Thus, this study supports that the IFN signaling pathway in conjunction with the BER pathway have an important role in viral clearance and persistence. Importantly, less than 30% of chronically HBV infected patients respond to IFN-α treatment [18], raising the question whether IFN non-responders have “defects” in the IFN signaling pathway.

In addition to the hepatocyte-specific effects of IFN signaling, HBV-specific T cells secrete IFN-γ and TNF-α [63]. Increased serum levels of IFN-γ and TNF-α were quantified during acute HBV infection vs. chronic infection. These results indicate that HBV-specific T cells, via cytokine secretion, can reduce the levels of cccDNA in infected hepatocytes via APOBEC-mediated deamination and subsequent cccDNA decay [63]. APOBEC3 can be induced by IFN-α, IFN-γ, TNF-α and lymphotoxin β-receptor activation [60, 64]. Indeed, recent studies have demonstrated direct antiviral properties of Toll-Like-Receptor (TLR) agonists in suppressing HBV replication in hepatocytes [65].

HBV-associated HCC and IFN signaling:

IFNs can mediate both antiviral and anti-proliferative effects [66]. ISGs expressed in response to IFN-α therapy mediate diverse biological functions including anti-tumor activity [67]. Thus, adjuvant IFN-α therapy is used for the treatment of specific types of cancers [68–70] and also for hepatocellular carcinoma [71]. Unfortunately, many HCC patients do not respond to treatment with IFN-α [18]. The study by Yang et al [72] compared ISG expression between the IFN-α responsive vs. non-responsive patients with HCC and HBV-related HCC, and identified the differential expression of the ISG IFIT3 between the two groups. The higher expression of IFIT3, in comparison to the other IFIT genes (IFIT1, IFIT2 or IFIT5), predicted better response to IFN-α therapy. Accordingly, it was proposed that IFIT3 could serve as a biomarker for predicting IFN-α responsiveness of HCC patients [72]. IFIT3, also known as RIG-G, is a direct transcription target of STAT1 [73]. Since expression of IFIT3 as well as of the other IFTs, is significantly reduced in liver tumors from IFN-α non-responders, one would predict defects in the mechanism(s) activating STAT1. For example, as described earlier, RIG-I promotes the nuclear translocation of activated STAT1 by disrupting STAT1 interaction with its negative regulator SHP-1 [56]. Importantly, RIG-I is severely downregulated in HCC, including poor prognosis HBV-related HCC [56]. Whether RIG-1 expression is also reduced in the IFN-α non-responders has not been investigated. Certainly, deregulation of various mechanisms that are functional in the context of HBV infection [43, 51] could mediate loss of STAT1 activation (Fig. 4).

Figure 4:

Mechanisms that regulate STAT1 function in HBV infected cells. Molecules (PRMT1, SETD2, RIG-I) depicted by green boxes facilitate STAT1 activation, whereas those in red boxes (PP2A, SHP-1, RNF2) are negative regulators of STAT1 activity.

Another possibility is that poor prognosis HCCs from IFN-α non-responders [72] lack transcription of STAT1. Interestingly, our studies (Andrisani et al unpublished data), presented at the 2^nd International Conference on Cytokine Signaling in Cancer, reported that reduced transcription of STAT1 maybe a feature of hepatic cancer stem cells (hCSCs). We have identified a cellular mechanism hijacked by HBV and linked to poor prognosis HBV-associated HCC [74–76]. This novel mechanism involves the chromatin modifying PRC2 complex, RNA helicase DDX5, and lncRNA HOTAIR. PRC2 [77] mediates repressive histone modifications (H3K27me3), while the RNA helicase DDX5 stabilizes/activates the HOTAIR/PRC2 complex and inhibits expression of pluripotency genes. HBV infection downregulates DDX5 [74–76]. Moreover, HBV-related HCCs associated with poor prognosis exhibit reduced expression of DDX5, supporting the clinical relevance of this mechanism [75]. We have also demonstrated [78] that loss of PRC2 function in a subpopulation of HBV replicating cells is associated with enhanced expression of pluripotency genes and hCSC genes. Interestingly, this subpopulation of HBV replicating cells also exhibits Wnt activation and resistance to chemotherapeutic agents, suggesting that these cells have CSC properties. Employing RNAseq analyses we mapped the transcriptome of these HBV replicating cells with hCSCs properties and Wnt activation. To our surprise, we observed reduced expression of interferon signaling genes, including STAT1, only in HBV replicating cells with hCSC properties/Wnt activation (Andrisani et al, Unpublished data). It is well established that in the context of oncogenesis, STAT1 can be either pro-apoptotic and anti-immunity, or pro-oncogenic [79]. However, what determines these opposing functions of STAT1 is not yet understood [79]. Cell intrinsic changes, for example the cellular status of epigenetic chromatin modifiers, e.g., the DDX5/HOTAIR/PRC2 complex, influenced by HBV infection [75], could, in turn, influence the function of STAT1 as oncogenic vs. pro-apoptotic.

Loss of IFN signaling in aggressive tumors and cancer stem cells (CSCs):

Accumulating evidence from the study of other cancer types supports the hypothesis that loss of IFN signaling is linked to aggressive/metastatic cancer and CSCs, as discussed below.

In a subset of metastatic prostate tumors both the INFGR1 gene and its downstream genes were found to be downregulated [80]. Interestingly, this subset of metastatic tumors exhibited enhanced expression of c-MYC and of the essential PRC2 subunit EZH2. It was shown that in MYC-driven prostate cancer the INFGR1 promoter is the direct target of EZH2. Importantly, EZH2 knockdown restores IFNGR1 expression and sensitizes cells to IFN-γ treatment [80]. In another study of metastatic melanoma, loss of IFN-γ pathway genes was identified as a mechanism of resistance in anti-CTLA-4 (ipilimumab) therapy [81]. They observed that cell lines generated from patient-derived primary tumors that were ipilimumab-resistant melanomas contained genomic mutations in IFN-γ pathway genes [81]. Also, recent studies with glioblastoma multiforme (GBM), the most lethal/malignant brain tumor, demonstrated loss of function/inhibition of the IFN regulatory factor 3 (IRF3). GBM tumors are characterized by remarkable proliferation, chemo-resistance, and invasion capacity. Significantly, the observed loss of function of IRF3 was linked to the invasion potential of GMB, through its surrounding neural tissue. IRF3 represses transcription of pro-invasive extracellular matrix (ECM) proteins [82]. It is unknown whether GBMs express reduced levels of IRF3, or whether inactivating mutations are associated with loss of IRF3 function. Another study from Yibin Kang’s laboratory recently demonstrated that CSCs exhibit the same properties as normal stem cells in evading IFN responses and cellular differentiation [83]. Specifically, they demonstrated that increased expression of the microRNA miR-199a both in normal mammary stem cells and breast CSCs targets the downregulation of the nuclear corepressor LCOR which sensitizes cells to IFN-induced differentiation and senescence. More recent studies in diffuse large B cell lymphoma (DLBCL) identified the targets of the constitutively activated STAT3, employing whole transcriptome analysis [84]. Interestingly, they found that STAT3 not only activates multiple oncogenic pathways, but STAT3 negatively regulates the IFN signaling pathway by suppressing transcription of STAT1, STAT2, IRF7 and IRF9 [84].

In terms of HCC and HBV-associated HCC the significance of downregulation of IFN signaling and STAT1, remains to be understood. IFNs act directly to inhibit tumor cell growth, and indirectly to activate anti-tumor immunity. Interestingly, Her2/neu transgenic mice carrying a nonfunctional mutation of INFAR1, exhibited earlier onset and increased tumor formation [85]. Significantly, these tumors displayed enhanced expression of the breast cancer stem cell marker ALDH1A1. These studies demonstrated that disruption of IFN signaling promotes Her2/neu tumor progression and formation of CSCs [85]. Whether, the transcriptional downregulation of STAT1 that we observed in the subset of HBV replicating cells with Wnt activation, is indicative of the hCSC phenotype is currently under investigation (Andrisani et al unpublished results). Interestingly, recent studies from C. Rice’s laboratory demonstrated that normal stem cells are resistant to viral infection because there is an intrinsic expression of a subset of ISGs that confers protection from viral infection, and that only upon differentiation the cells assume inducible IFN expression of ISGs [86]. Thus, the transcriptional repression of STAT1 that we have observed in a subset of Wnt-positive HBV replicating cells (Andrisani et al unpublished data), may be linked to a hCSC phenotype.

Concluding Remarks

HBV has evolved mechanisms to inhibit IFN signaling during the phases of acute and chronic infection, and during the process of hepatocyte transformation. These mechanisms, described herein, focus on the essential IFN signaling transcription factor STAT1 (Figs. 3–4). While during acute infection HBV inhibits the activity and nuclear translocation of phospho-STAT1 [15, 16, 22, 27], intrinsic changes, i.e., cellular reprogramming of the HBV transformed hepatocyte may also target the transcriptional repression of STAT1, associated with a hCSC phenotype. Ongoing studies address this hypothesis.

Abbreviations:

HBV

Hepatitis B Virus

NTCP

Sodium Taurocholate Co-transporting Polypeptide

cccDNA

covalently closed circular DNA

pgRNA

pregenomic RNA

IFN

Interferon

ISGs

Interferon Stimulated Genes

STAT

Signal Transducer and Activator of Transcription

BER

Base Excision Repair

PRC2

Polycomb Repressive Complex 2

RNAseq

RNA sequencing

HCC

Hepatocellular Carcinoma

hCSC

hepatic Cancer Stem Cell

GBM

glioblastoma

DLBCL

diffuse large B cell lymphoma