Hepatitis B Virus X gene in Hepatocellular Carcinoma

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A Viral Biorealm page on the family Hepatitis B Virus X gene in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is among the most frequently diagnosed cancers worldwide. For over forty years physicians and researchers have observed an association between HBV infection and development of HCC. Of the seven HBV genes, hepatitis b virus x protein (HBx, 17.5 kDa) is the smallest and required extensive efforts to characterize; its elusiveness is highlighted by its designation “X” [1]. The protein functions as a transcriptional transactivator of both viral and cellular transcripts, in addition inducing cytoplasmic signal transduction pathways [1]. Interestingly, HBx was a more potent inducer of transcription upon deletion of its N-terminal 1 to 50-amino-acid fragment, suggesting this domain may function as a negative regulatory element [1].

Figure 1. text. [cite].





Virus Overview

Hepatitis B virus is transmitted by mucosal or percutaneous contact with body fluids or blood from an infected individual the blood or body fluids of an infected person [18]. This occurs most often through injection-drug use, sexual contact with an infected person, or mother-to-child transmission [18]. The infection localizes in the liver and has been linked to the development of several diseases including cirrhosis and hepatocellular carcinoma. The hepatitis B vaccine contains hepatitis B surface antigen (HBsAg) and is administered in a course of three injections. The vaccine induces serum levels of anti-HBsAg sufficient enough to activate the recruitment cytotoxic T cell and NK cell responses to viral infection such that the virus is wiped out…

Hepatitis B Virus Genome

The hepadnaviral genome is packaged as relaxed circular (RC), partially double-stranded DNA about 3.2 kb in length. After viral DNA enters the nucleus, the partially double-stranded viral DNA is repaired and the (+) DNA strand is extended, rendering a molecule of functional, covalently closed circular DNA (cccDNA). The HBV genome consists of four genes with four partially-overlapping ORFs: core (C), polymerase (P), surface antigen (S), and X. The core gene consists of the precore and core regions [17]. The presence of several in-frame translation initiation codons for S and C genes allows for the translation of related but functionally distinct proteins [17]. The C ORF encodes the viral nucleocapsid (HBcAg) at the core ORF and hepatitis B e antigen (HBeAg) at the precore ORF. The S ORF codes for envelope proteins, HBsAg [17].

Figure 1. text. [cite].

Polymerase (pol) is a large protein and consists of three subunits: a reverse transcriptase, a terminal protein subunit involved in (-) strand synthesis, and the ribonuclease H which helps replication and breaks down pregenomic RNA [17]. Strand-specific synthesis during replication is controlled by two genomic elements, direct repeats 1 and 2 (DR1 and DR2) in the 5’ ends of the plus strand. Liver-specific expression of viral gene products is controlled by two viral enhancer elements, En1 and En2.

Replication Cycle

To enter a cell, hepatitis B virus binds an unidentified receptor and is endocytosed. Studies using duck HBV models indicate that carboxypeptidase D plays an essential role in viral entry [17]. After uncoating of the nucleocapsid core, the viral genome enters the nucleus where the single-stranded gap region is repaired by viral polymerase, leaving the viral genome as cccDNA, the functional template for transcription by host RNA polymerase II. The fact that HBV mRNAs are subject to alternative splicing presents a challenge in the development of small molecule inhibitors targeting a specific viral binding domain [19]. Viral mRNA is transcribed as both pre-genomic and pre-core mRNA. Pre-genomic mRNA (pgRNA) serves as the template for reverse transcription and translation of pol and core; pre-core mRNA levels determine expression of the pre-core protein product. As viral proteins are synthesized, the core packages pgRNA in response to a cis-acting stem loop denoted epsilon. After encapsidation, pgRNA is reverse transcribed by pol to partially double stranded relaxed circular (RC) DNA. After interacting with proteins along the endoplasmic reticulum to become a mature virion, HBV lyses the cell membrane and is released into the extracellular environment.


Hepatitis B Virus Protein X Interacts with Host and Viral Components

Since the first report of a miRNA possessing antiviral properties in 2005 [15], researchers have begun to explore the possibility of interactions between host miRNAs and HBV. These interactions have provided mechanistic insights regarding the role of HBx in host gene expression and HCC development in HBV-infected individuals.


In vitro, HBx binds eukaryotic basal transcription factors essential for proper RNAP II-DNA interactions and promoter escape, including TATA-binding protein (TBP), TFIIB, TFIIH, and the RNAP subunit RPB5 [1, 11]. These interactions facilitate transcription of viral genes. HBx has been shown to suppress apoptosis of hepatocytes via induction of survivin, disruption of the tumor suppressor p53, and activation of c-jun N-terminal kinases [13, 12]. In infected cells, HBx induces overexpression of a cellular target, HBXIP, which functions as a regulator of centrosome dynamics and cytokinesis – critical aspects of mitosis [14]. Furthermore, it was reported that HBx activates src and the ras/raf/ERK (extracellular signal-regulated kinase) pathway, which leads to proliferation of quiescent cells [12].


       There have been numerous studies investigating the mechanisms of HBx-mediated cell proliferation and HCC development. Two studies in the 1990s demonstrated HBx potentiates c-myc induced liver oncogenesis in a mouse model [3, 4]. HBx mediates the expression of a wide variety of host RNAs and genes, including transcription factors, the p-53 tumor-suppressor gene, oncogenes, ribosomal protein S27a [6], and ribosomal RNAs [7]. In HCC cells, overexpression of HBx and the oncogene c-myc each increased rRNA levels; when cells were co-transfected with HBx and c-myc, an additive effect on both rRNA synthesis and cell division was observed [7]. Furthermore, in vitro analyses of HCC cells infected with HBx-knockdown HBV demonstrate the existence of particular HBx-dependent proliferative signaling events in this cancer [5]. 


According to Kumar et al., HBx is also significantly involved in evasion of the innate immune response. The authors found that HBx binds an adaptor protein called IPS-1 that mediates IFN-β release. HBx-IPS-1 binding decreased the ability of cells to produce and secrete active IFN-β. In the context of immune system evasion, HBV has evolved much like how a tumor evolves. Collectively, these findings suggest that HBx is a key mediator of HCC development in HBV infected individuals.

Overview of Interactions between HBV and Host miRNAs

Figure 1. Screening for miRNAs involved in HBV expression. This step follows an initial in silico screen. [Hu et al. 2012].

Several cellular transcription factors have an affinity for HBV enhancer elements and thus are crucial for efficient viral replication. A 2011 review reported C/EBP and CREB as TFs necessary for efficient viral replication. C/EBP is “CAAT enhancer-binding protein,” designated as such because of its affinity for the CAAT promoter motif, and CREB is cAMP-response element binding protein. Since publication of the review, Hu et al. reported peroxisome proliferator-activated receptor alpha (PPARA) as a host TF that binds viral enhancer elements and induces transcription of viral genes. A common method to screen for host miRNAs with HBV mRNA sequence alignment is to conduct an in silico high-throughput screen. The miRNA that targets PPARA (miRNA-141) was selected in a preliminary screening of “hit” sequences because it was the most effective inhibitor of HBV replication in a screen of 64 miRNA sequences.

Figure 1. text. [cite].


In an effort to elucidate the precise host-virus interactions which result in HBx-regulated miRNA expression, Ren et al. overexpressed HBc, HBs, and HBc, and HBx proteins separately in the presence of overexpressed Drosha in HCC cells. They observed decreased Drosha activity only in the presence of HBx. Inhibition of HBx by shRNA knockdown corresponded with increased Drosha activity. Considering Drosha is the key protein involved in processing functional miRNAs, this study provides strong evidence that HBx alters host gene expression through a complex network of interactions. Ren et al. provided a base of support for further study of HBx-miRNA interactions in HCC development. In 2011, Wu et al. showed that HBx down-regulates the miR-16 family in malignant hepatocytes. This family of RNAs targets various cyclins that are responsible for activating cyclin-dependent kinases, making these miRNAs key regulators of the G1/S cell-cycle checkpoint [2]. Also in 2011, Potenza et al. demonstrated that has-miR-125a-5p, a microRNA expressed in human liver, down-regulates the expression of HBV S gene and, the corollary, the amount of secreted HBsAg. The authors speculate that the in vivo effect of introducing this miRNA to localized sites would be decreased viral load as it has been shown that experimental knockdown of HBsAg leads to suppression of HBV replication [Human microRNA hsa-miR-125-5p interferes… ].

X. Wei et al. (2012) investigated HBx-mediated expression of a miRNA (miR-101) that targets DNA methyltransferase DNAMT3A [8]. The ability of HBx to down-regulate activity of a DNA methyltransferase suggests hepatitis B virus can influence epigenetic modifications to alter host gene expression. Evidence of miR-101 down-regulation in HBV-positive HCC tumor tissue suggests that miR-101 plays a tumor-suppressive role in HCC development [8]. The authors reported DNAMT3A targets several tumor-suppressor genes including RASSF1, APC, and CDKN2A. To summarize, HBx mediates increased expression of DNMT3A by decreasing miR-101 levels, thus decreasing expression of tumor-suppressor genes like RASSF1, APC, and CDKN2A. It is likely that these events contribute to the development of HCC in HBV infected individuals.

Current and Potential HBV Therapies

The seven drugs currently approved for the treatment of chronic hepatitis B in the US and Europe include IFN-α, pegylated IFN-α, and nucleotide analogues (NUCs) lamivudine, adefovir, entecavir, telbuvudine, and tenofovir [10]. In general, interferons are capable of inhibiting viral replication and stimulating an anti-viral immune response (mobilizing NK cells activity). NUCs inhibit synthesis of the viral genome by HBV polymerase by competing with natural NTPs for polymerase uptake. The lack of a hydroxyl group on NUCs prevents polymerase from forming an additional covalent bond, effectively halting DNA synthesis. Tenofovir, an adenosine analogue, is a common component of HAART regimens used to treat HIV infected individuals.


Despite extensive efforts to characterize HBx, a successful inhibitor of the viral protein has not been developed. However, according to a 2012 issue of HBV Journal Review, researchers at UC-Boulder have identified two small proteins that help inhibit cell death, Bcl-2 and Bcl-xL, as the “bona fide” host targets of HBx [9]. The group determined that a result of HBx interaction with these proteins is increased intracellular calcium levels, which then triggers activation of necrosis in C. elegans through Ca2+ dependent proteases [18]. Via utilization of the genomically simple C. elegans model, researchers hope to develop drugs that bind the Bcl-2/Bcl-xL recognition motif of HBx to reverse the negative effects of the viral oncoprotein without disrupting the normal function of Bcl-2/Bcl-xL.


Further research could be aimed at designing ways to restore miRNA functions (depending on the case) and especially at designing HBx inhibitors. Shim and Kwon (2010) published a review of methods to locally deliver short RNAs in vivo. Theoretically, this type of protocol may be adopted to knockdown HBx expression in hepatocytes or to increase the intracellular concentrations of tumor-suppressive miRNAs that HBx down-regulates (i.e. miR-101, miR-16).

Wang et al. (2012) utilized the ability of HBx to induce the expression of HCC-related genes to develop a library of biomarkers to characterize the stage and role of HBV in patients with HCC.

References

[1] Bouchard, MJ, and RJ Schneider. "The Enigmatic X gene of Hepatitis B Virus."Journal of Virology. 78.23 (2004): 12725-12734.

[2] Wu, G, F Yu, et al. "Hepatitis B virus protein downregulates expression of the miR-16 family in malignant hepatocytes in vitro." British Journal of Cancer. 105. (2011): 146-153.

[3] Kim CM, Koike K, Saito I, Miyamura T, Jay G. “HBx gene of hepatitis B virus induces liver cancer in transgenic mice.” Nature. 1991;351

[4] Terradillos O, Billet O, Renard CA, Levy R, Molina T, Briand P, Buendia MA. The hepatitis B virus X gene potentiates c-myc-induced liver oncogenesis in transgenic mice. Oncogene. 1997;14 (4:395–404.

[5] He, Yan, Hui-qing Sun, et al. "Knockdown of HBx by RNAi inhibits proliferation and enhances chemotherapy-induced apoptosis in hepatocellular carcinoma cells." Medical Oncology. 27. (2009): 1227-1233.

[6] Fatima , G, G Mathan, et al. " The HBx protein of hepatitis B virus regulates the expression, intracellular distribution and functions of ribosomal protein S27a." Journal of General Virology. (2011): 706-715.

[7] Shukla, SK, and V Kumar. "Hepatitis B virus X protein and c-Myc cooperate in the upregulation of ribosome biogenesis and in cellular transformation." FEBS Journal. (2012): 3859-3871.

[8] Wei, Xufu, and Tingziu Xiang . "miR-101 is down-regulated by the hepatitis B virus protein and induces aberrant DNA methylation by targeting DNA methyltransferase 3A." Cellular Signaling . (2012)

[9] Kukka, Christine. “Researchers Uncover New Ways to Prevent Viral Replication" HBV Journal Review. 9.11 (2012): 2-3

[10] Grimm, Daniel, Robert Thimme, et al. "HBV life cycle and novel drug targets.” International Journal of Hepatology. 5. (2012): 644-653.

[11] Qadri, I, H Maguire, et al. "Hepatitis B virus transactivator protein X interacts with the TATA-binding protein." Proceedings of the National Academy of Sciences. 92. (1995): 1003-1007.

[12] YC, Kim, and Song KS. " Activated ras oncogene collaborates with HBx gene of hepatitis B virus to transform cells by suppressing HBx-mediated apoptosis." Oncogene. 20. (2001): 16-23.

[13] Marusawa, H, S Matsuzawa, et al. "HBXIP functions as a cofactor of survivin in apoptosis suppression." EMBO Journal. 22.11 (2003): 2729-2740.

[14] Fujii, Ryoji, Changjun Zhu, et al. " HBXIP, Cellular Target of Hepatitis B Virus Oncoprotein, Is a Regulator of Centrosome Dynamics and Cytokinesis." Journal of Cancer Research. (2006)


[15] Lecellier, CH, P Dunoyer, et al. “A cellular microRNA mediates antiviral defense in human cells.” Science. 308. (2005): 557-560

[16] Ren, Min, Dongdong Qin, et al. "Correlation between hepatitis B virus protein and microRNA processor Drosha in cells expressing HBV." Antiviral Research. 94. (2012): 225-231.

[17] Liang, TJ. “Hepatitis B: The Virus and Disease." Hepatology. 49. (2009): S13-S21.

[18] Division of Viral Hepatitis. United States. Center for Disease Control. Viral Hepatitis Surveillance. 2010

[18] Geng, Xin, Brian L. Harry, et al. “Hepatitis B virus X protein targets the Bcl-2 protein CED-9 to induce intracellular Ca2+ increase and cell death in Caenorhabditis elegans." PNAS. 109.45 (2012): 18465-18470.

[19] Lee, GH, S Wasser, et al. " Hepatitis B pregenomic RNA splicing—The products, the regulatory mechanisms and its biological significance." Virus Research. 136.1-2 (2008): 1-7

[20] Wang, Wen, LJ Zhao, et al. “Application of HBx-induced anti-URGs as early warning biomarker of cirrhosis and HCC." Cancer Biomarkers. (2012): 29-39.


Beck, Juergen, and Michael Nassal. "Hepatitis B virus replication." World Journal of Gastroenterology. 13.1 (2007): 48-64.

Cheng, Alfred A.L., Jun Yu, et al. "COX-2 mediates hepatitis B virus X protein abrogation of p53-induced apoptosis."Biochemical and Biophysical Research Communications. (2008)

Liu, Wan-Hsin, Shiou-Hwei Yeh, et al. "Role of microRNAs in hepatitis B virus replication and pathogenesis." Biochemica et Biophysica Acta. (2011): 678-685.

Hepatitis B Virus Regulatory HBx Protein Binds to Adaptor Protein IPS-1 and Inhibits the Activation of Beta Interferon