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 and is capable of inducing cytoplasmic signal transduction pathways [1]. HBx has been shown to interact directly with a wide variety of biomolecules, including ssDNA, multiple transcription factors (TFs), proteins associated with apoptosis, TATA-binding protein, and an essential subunit of human RNA polymerase I, II, and III, RPB5. Interestingly, HBx was a more potent activator 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. HBV-infected individuals are more vulnerable to develop cirrhotic liver and hepatocellular carcinoma [www.livercancerfree.org/livercancer.php].

Virus Classification

Family: Hepadnaviridae

Genus: Orthohepadnavirus

Baltimore Classification: Class VII - double-stranded DNA viruses that replicate through a single-stranded RNA intermediate

Genome: partially double-stranded DNA, ~3.5 kb

Virus Overview

Hepatitis B virus is transmitted by mucosal or percutaneous contact with body fluids or blood from an infected individual [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 of the liver 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 areas of viral infection and wipe out the pathogen.

Hepatitis B Virus Genome

The hepadnaviral genome is packaged as relaxed circular (RC), partially double-stranded DNA about 3.5 kb in length. The (+) strand exists bound to DNA-binding proteins which inhibit its complete synthesis. In the nucleus, these proteins dissociate and viral polymerase extends the (+) strand, leaving the viral genome as covalently closed circular DNA (cccDNA). The virus benefits from a cccDNA genome because the DNA strand stores energy in supercoils and, as a result, is less reliant on host ATP-dependent helicase activity for DNA replication.

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 the surface antigen, HBsAg [17].

Figure 2. Schematic of Hepatitis B virus genome. Though relatively small, the X gene is the most difficult to characterize. [Beck J et al. 2006].

Polymerase (pol) is a large protein and consists of three subunits: a reverse transcriptase, a terminal protein subunit which provides the active site for DNA 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 [17].

Replication Cycle

To enter a cell, hepatitis B virus binds an unidentified receptor and is endocytosed. Utilizing duck HBV as a model infection system, several blotting techniques, and immunofluorescence microscopy, Breiner et al. (1998) showed that carboxypeptidase D (gp180), a cell surface receptor protein involved in intracellular trafficking to the Golgi, plays a role in viral entry via association with hepatitis B pre-S polypeptide [21].

Figure 3. Schematic of the Hepatitis B virus replication cycle [10].

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 specific viral binding domains [19].

Viral DNA is transcribed and the resulting mRNA may be translated into a protein or saved as pre-genomic information. 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. The immature virion then interacts with proteins along the endoplasmic reticulum where it matures prior to lysing the cell membrane, triggering its release into the extracellular environment.

Hepatitis B Virus Protein X Interacts with Host and Viral Components

There have been numerous studies investigating the mechanisms of HBx-mediated cell proliferation and HCC development. Two studies in the 1990s demonstrated that 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 p53 tumor suppressor gene, oncogenes, ribosomal protein S27a [6], ribosomal RNAs [7], miRNAs, and proteins responsible for cell cycle regulation. 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 demonstrated the existence of particular HBx-dependent proliferative signaling events in this cancer [5].

Figure 5. "GST-resin pull-down assay using glutathione-Sepharose resin pre-bound to GST-fused full-length or truncated RPB5 protein. The resin was incubated and washed. Lanes 3, 5, 7, 9, and 11, input is HBx protein alone (100 ng); lanes 4, 6, 8, 10, and 12, input is HBx (100 ng) together with TFIIB (100 ng). Lanes 1 and 2, 10% of input of HBx or HBx/TFIIB, respectively." [25]. Follow the link for a thorough description of a GST-resin pull-down assay: http://www.piercenet.com/Objects/View.cfm?type=ProductFamily&ID=005C9A96-1498-4B7B-B02A-F290370E75C2

In 1996, it was confirmed by electromobility shift assays (EMSA) that HBx binds single-stranded DNA in a manner that is non-sequence-specific (Fig 4) [24]. A year later, researchers used GST-resin pull-down assays, far-Western blotting, and in vivo co-immunoprecipitation to show that HBx is capable of forming a trimeric complex with RPB5 and TFIIB, a general TF that facilitates transcription by interacting with the pre-initiation complex (Fig 5) [25]. A separate group used similar techniques to show interactions between TATA-binding protein and HBx. Collectively, these findings suggest that HBx enhances transcription efficiency of viral and host genes. Researchers moved outside the realm of general transcription events (RPB5, TBP, TFIIH) and looked for interactions between HBx and HCC-related signaling events.

Figure 4. "HBx binds to the single-stranded region of native HBV DNA. EMSA of HBx-ssDNA interactions in the presence of a phenol-extracted genomic HBV and filled-in DNA is shown. Lane 1, free probe; lane 2, GST; lanes 3–8, GST-X. Lanes 4–8, in the presence of competitor HBV native DNA (lane 4); HBV filled-in DNA (lane 5); M13 ssDNA (lane 6), and M13 filled-in DNA (lane 7). HBV and M13 DNAs were filled in with reverse transcriptase, and then 0.1 units of mung bean nuclease was added to remove any residual unfilled ssDNA. 10 ng of HBV and M13 DNAs were added as competitor." [24].
Figure 6. "UV cross-linking of HBx-ssDNA. Lane 1, no protein; lane 2, GST; lanes 3–6, GST-X. This is shown in the presence of increasing amounts (26, 52, and 260 nM;lanes 4–6, respectively) of E. coli SSB. Lanes 3–6 contain 91 nM GST-X" [24].
Figure 7. To identify whether HBx interacts with TFs at promoters of target genes, Sung et al. used chromatin immunoprecipitation with antibodies against HBx and primers specific for each promoter region of interest. [Sung W et al. 2009].

HBx was shown to suppress apoptosis of hepatocytes via inducing survivin, disrupting the p53 gene, and activating c-jun N-terminal kinases [13, 12]. Each of these proteins have crucial roles in regulating the cell cycle; a hallmark of tumorigenesis is mutation of the p53 gene.

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]. Additionally, Kumar et al. demonstrated that HBx is significantly involved in evasion of the innate immune response [30]. The authors found that HBx binds a protein (IPS-1) capable of mediating 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.

More recently, Sung et al. (2009) performed the first ChIP-on-chip analysis of HBX-host interactions [22]. The core hypothesis of the study, summarized in Fig 6, was that HBx directly binds transcription factors that target genes involved in cancer development. The group first showed that HBx co-immunoprecipitates with transcription factors E2F1, SMAD4, and YY1. These TFs activate target genes involved in regulation of the cell cycle and apoptosis. It was shown that HBx interacts with the promoter of five target genes using anti-HBx antibodies and primers specific for the selected promoter regions. To verify that E2F1, SMAD4, and YY1 directly regulate transcription of target genes, the three TFs were knocked-down with siRNA and the transcript levels of their respective target genes were determined by RT-qPCR. Indeed, the experiment verified that each TF is responsible for regulating mRNA levels of target genes, and HBx has an affinity for these TFs which regulate cancer development. The authors plugged their biological finding into the REACTOME database using the NextBio search engine to identify "core pathways that may be deregulated by HBx through its binding to the promoters of target genes." Six pathways were identified, including the Jak-STAT signaling pathway. This is especially interesting in light of a body of literature which identifies the Jak-STAT pathway as overactive in individuals with HCC. This notion is highlighted in an article which appeared in Gastroenterology, "Ubiquitous Activation of Ras and Jak/Stat Pathways in Human HCC," (Calvisi, et al., 2006).

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 8. HBx down-regulates expression of host miRNAs. Many of these miRNAs target transcription factors capable of binding viral enhancer elements and increasing the rate of viral assembly [Liu et al. 2011]. For the virus to take complete advantage of host transcriptional machinery, miRNAs which inhibit that machinery should be down-regulated. It is thought that HBx has evolved to down-regulated these miRNAs. If the host uses miRNAs as a mechanism of battling infection by decreasing the rate of virus replication, then HBx is the viral counterattack. [29]
Figure 9. Intracellular processing of miRNAs. After a pre-miRNA is transcribed, Drosha "cuts" the short stem loop from the RNA polymer. [16].

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.

Figure 10. The ability of 64 different host miRNAs to inhibit HBV replication was determined by ELISA specific for HBsAg. [Hu et al. 2012].

Several cellular transcription factors have an affinity for HBV enhancer elements and thus are crucial for efficient viral replication (Fig 8). 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 another host TF that binds viral enhancer elements and induces transcription of viral genes.

HBx-miRNA interactions are identified by several techniques. Hu et al. (2011) were interested in identifying a host miRNA that is capable of regulating HBV replication. The initial screen they employed was a three step process. First, the group selected 64 miRNAs from an expression microarray that are known to be involved in cell differentiation, viral infection, and cancer. For each miRNA, they co-transfected HCC cells with HBV DNA plasmid + miRNA or control plasmid + miRNA. Then, HBsAg levels for each of the 64 cell populations were assayed by ELISA to determine the ability of each miRNA to inhibit HBV replication (Fig 9).

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 found decreased Drosha activity only in the presence of HBx. Inhibition of HBx by shRNA knockdown corresponded with increased Drosha activity. Considering Drosha is a key protein in the processing of 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 hepatocytes, down-regulates the expression of HBV S gene and, the corollary, levels of secreted HBsAg. The authors speculate that the in vivo effect of introducing this miRNA to localized sites would be decreased viral load. It has been shown that experimental knockdown of HBsAg leads to suppression of HBV replication [23].

Figure 11. Analysis of miR-101 and DNMT3A mRNA levels in HBV or HBx transfected cells by RT-qPCR (A) HCC cells transfected with total HBV DNA (HEPG2.2.15) express fewer miR-101 molecules than untransfected HEPG2 cells. (B) miR-101 is significantly down-regulated in HEPG2 cells overexpressing HBx relative to cells over-expressing GFP. (C) DNMT3A is up-regulated in HEP2.2.15 cells relative to HEPG2 cells. (D) DNMT3A is up-regulated in HEPG2 cells over-expressing HBx relative to HEPG2 cells over-expressing GFP. [Wei et al. 2012].

X. Wei et al. (2012) investigated HBx-mediated expression of a miRNA (miR-101) that targets DNA methyltransferase DNAMT3A (Fig 11) [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 like 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 (Fig 12). It is likely that these events contribute to the development of HCC in HBV infected individuals.

Figure 12. (A, B) qRT-PCR analysis demonstrating inhibition of miR-101 rescues DNMT3A-mediated transcriptional silencing of tumor-suppressor transcripts. (C) methylation-specific PCR of tumor-suppressor transcripts derived from HCC cells transfected with total hepatitis B virus DNA (HepG2.2.15) and exposed to miR-101 mimics or uninfected HCC cells (HepG2) exposed to miR-101 inhibitor. An increase in methylated CDKN2A in HepG2.2.15 cells suggests that a viral protein (HBx) downregulated levels of introduced miR-101 mimics, resulting in more methylation of the gene targeted by DNMT3A. [Wei et al. 2012].

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.

Figure 13. "The inhibitory effects on HBV RNA levels by transfection with HBV ShRNAs. NOD SCID mice were cotransfected with 12 μg pTHBV2 and 1.6-2.0 ml pWPT-HBV- ShRNA-1, 2 or negative control shRNA. The mice were sacrificed 4 d and 7 d after cotransfection, and hepatocytes RNA was extracted. Each experiment was carried out in triplicate. (A) The total levels of HBV S-mRNA in NOD SCID mouse livers were significantly reduced on day 4 or 7 after treatment with pWPT- HBV-shRNA-1 and shRNA-2 lentivirus. (B) The total levels of HBV C-mRNA were significantly decreased on day 4 or 7 after treatment with pWPT-HBV-shRNAs in mice. (error bars indicate standard errors). The values shown were the average of three experiments (means standard deviation). The mean value of empty control was defined as 100%. * P < 0.05 vs. the empty vector group. # P > 0.05 vs. the empty vector group." ].

Despite extensive efforts to characterize HBx, a successful inhibitor of the viral protein has not been developed. Crystal structures of HBx bound to target TFs would be useful in developing small molecule inhibitors via rational drug design. For example, 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 triggers necrosis in C. elegans through Ca2+ dependent proteases [18]. By utilizing 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.

In 2009, Deng et al. published promising results of an in vivo lentiviral therapy capable of decreasing viral load in mice [26]. The vector was cleverly designed to target the transcribed version of the HBV cccDNA genome. This pre-sliced RNA genome serves as both the immature template for translation and is stored as pre-genomic information. The authors used two shRNA duplexes "targeting the overlap region encoding the core antigen and the polymerase (pol)/surface antigen region (HBsAg) of the HBV genome" [26]. Decreased viral load in lentivirus-treated mice was confirmed by immunohistochemical staining, ELISA for HBsAg in serum, qPCR analysis of total HBV DNA in serum, and RT-qPCR analysis of HBV core-mRNA in serum (Fig 12).

In light of recent research regarding the role of miRNAs involved in HBV-related HCC, future studies could aim towards developing methods to restore miRNA functions in cases where HBx down-regulates these important and potentially antiviral sequences. It is possible that restoring proper miRNA function in HBV infected individuals will reduce their risk of developing HCC and even decrease the tumor burden in already-infected individuals. If Drosha function is indeed significantly inhibited in the presence of HBx [16], then researchers could investigate a method of restoring the activity of this crucial enzyme.


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