HEPATITIS B VIRUS: X GENE NAAZ ABBAS AND A. R. SHAKOORI. School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore.

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1 Proc. Pakistan Congr. Zool., Vol. 27, pp , HEPATITIS B VIRUS: X GENE NAAZ ABBAS AND A. R. SHAKOORI School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore. Content Abstract Hepatitis B Virus Liver injury by HBV Replication cycle Transmission Virale genome X gene of HBV Promoters and Signal Regions Mutations in the X gene HBx is important for the mammalian hepadnaviridae life cycle Role of X gene in carcinogenisis References Abstract.- With an estimate of 500 million people infected with the hepatitis B virus (HBV) worldwide, persistent HBV infection is maintained and linked to chronic hepatitis, cirrhosis, and development of liver cancer (hepatocellular carcinoma). The HBV nonstructural X protein, a key regulatory protein of the virus that is at the intersection of HBV infection, replication, pathogenesis, and possibly carcinogenesis. Mutations in the X region can involve the regulatory elements that control replication, such as the basal core promoter and Enhancer II. HBx is 154 amino acids in size, with a molecular mass of approximately 17.5 Kda. Keywords: Hepatitis B virus, HBV X gene, Role of X gene, HBxAg HEPATITIS B VIRUS The hepatitis B virus (HBV) is a unique, coated DNA virus belonging to the Hepadnaviridae family of viruses. HBV is not related to the hepatitis A virus or the hepatitis C virus. The HBV is also called the Dane Particle after its discoverer. These viruses primarily infect liver cells. Other viruses in this family can cause hepatitis in creation animals. These viruses include the woodchuck hepatitis virus, the ground squirrel hepatitis virus, and the duck HBV. Models have been developed to study the HBV and to evaluate new drugs to treat it

2 128 N. ABBAS AND A.R. SHAKOORI (Chen et al., 1993; Dandri et al., 1996). It is made up of the virus coat, the nucleocapsid and the viral DNA genome. The genes of the hepatitis B virus contain genetic codes to make a number of protein products, including hepatitis B surface antigen (HbeAg), hepatitis B core antigen (HbcAg), hepatitis B e antigen (HbeAg), and DNA polymerase. These four proteins are important because these are used as serologic (blood) markers specifically for HBV to diagnose hepatitis B viral infection. Certain markers can also be used to determine the infectivity (ability to infect others) and the activity of the HBV infection, which are related to the level of the virus in the blood. Hepatitis B is preventable. All children and adults at high risk for hepatitis B should be vaccinated. Passive immunization with specific immune globulin (HBIG) is also available to protect susceptible, exposed individuals. LIVER INJURY BY HBV The HBV itself does not directly cause damage to the liver. Rather, the body s immune (protective) response to the virus (a foreign material) paradoxically causes the damage. Thus, in a hepatitis B viral infection, the body s immune response to the virus is responsible for both the elimination of the HBV from the body and recovery from the infection. Accordingly, an acute hepatitis B viral infection can lead to recovery (the usual outcome), to acute liver failure (really), and sometimes to chronic infection. At some point, however, the chronic hepatitis can progress to cirrhosis (severe scarring, or fibrosis) of the liver. Finally liver cancer can develop in chronic HBV infected patients as a complication of advanced cirrhosis. The way in which the cancer develops is not fully understood. It is thought, however, that the HBV DNA somehow becomes incorporated into the liver cell DNA of the patient. REPLICATION CYCLE Like all viruses, HBV requires living cells in which to multiply. After penetration by the virus particle, its genetic material is released and insinuates into the nucleus of the host cell. Once inside the nucleus, the genetic material stimulates the host cell to produce new viral components. These are then assembled to new virus within the host cell during the replication phase. It is

3 HEPATITIS B VIRUS: X GENE 129 initially inaccessible to attack by the immune system. Despite this, some specific antigens such as the core antigen (HbcAg) pass to the surface of the hepatocytes, as a result of which the affected cells are then attacked by the immune system. TRANSMISSION HBV is spread or acquired through exposure to infected blood (for example, intravenous drug use) or the body s secretions. The highest concentrations of HBV are found in the blood, semen, vaginal discharge, breast milk, and saliva. There are only low concentrations of HBV in the urine and non in the faeces. Last (but not the least), HBV can spread from infected mothers to their babies at the time of birth (so-called vertical transmission), but not through food, water, or casual contact. VIRAL GENOME The genetic material of the HBV is partly single-stranded (RNA) and partly double-stranded (DNA). If infection occurs, then the single-stranded sequences in the cell nucleus of the host cell combine with the RNA of the host cell to form a double strand. The DNA of the HBV contains 3200 base pairs. The individual genes coded by the genetic material have been elucidated since discovery. The four overlapping open reading frames (ORF) contain the information for seven viral proteins through use of varying in-frame start codons (Fig. I). For example, the small hepatitis B surface protein is generated when a ribosome begins translation at the ATG at position 155 of the adw genome. The middle hepatitis B surface protein is generated when a ribosome begins at an upstream ATG at position 3211, resulting in the addition of 55 amino acids on to the 5 end of the protein. ORF P occupies the majority of the genome and encodes for the hepatitis B polymerase protein. ORF S encodes three surface proteins. ORF C encodes both the hepatitis e and core protein. ORF X encodes the hepatitis B X (HBx) protein that contains information for two proteins that function as activators to support transcription. However, the genome also contains genetic elements, which regulate levels of transcription, determine the site of polyadenylation, and even mark a specific transcript for encapsidation into the nucleocapsid (Wang et al., 1990; Haviv et al., 1998).

4 130 N. ABBAS AND A.R. SHAKOORI Fig. 1. A diagrammatic representation of the HBV genome. The inner circle represents the virion genomic DNA that is packaged within viral particles in the cytoplasm of infected cells, and the dashes indicate the region of the genome which is incompletely synthesized. The thick arrows represent open reading frames corresponding to core, envelope (surface antigen), polymerase (pol), and HBx proteins. The thin lines represent HBV RNAs. X GENE of HBV The exact function of HBx during HBV replication and its influence in HBV-associated hepatocellular carcinoma are still undefined, but common themes are emerging. Most studies agree that, if not absolutely essential for HBV infections, HBx at the very least plays a critical role in viral replication. HBx functions in the cytoplasm to activate various signaling pathways, many of which are controlled by modulation of cytosolic calcium. In the HBV genome, four long ORFs have been found that encode the virus core, surface and polymerase proteins as well as a protein examined contain two addition ORFs designated ORF5 and ORF6. Both ORFs are located in the X gene region, which

5 HEPATITIS B VIRUS: X GENE 131 corresponds to the 3 terminus of the linear RNA genome approximately 0.7 kb. ORF5 is located on the same strand as the four known viral genes and is codons in size. ORF6 is located on the DNA strand complementary to the one that encodes the other virus genes, and is approximately 210 codons in length. PROMOTERS AND SIGNAL REGIONS ORF X, which encodes a 17kd protein known as the HBx protein, has its own promoter controlling the transcription of a 0.9kb RNA. However, due to the proximity of the X promoter to enhancer I region, the precise borders remain controversial. The promoter is believed to lie within the region spanning nucleotides 1230 to The enhancer I element spans a region from nucleotides Many transcription factors are believed to interact with this region of the HBV genome. These molecules include HNF-3, HNF-4, EF-C and NF-1, to name a few. Both enhancer I and enhancer II can activate heterologous promoters despite their position or orientation. Deletion of either enhancer region results in a strong reduction of viral transcripts (Twu et al., 1987). Scientists have analyzed a series of plasmids in which the sequences located upstream from the HBV X gene were linked to the chloramphenicol acetyltransferase (CAT) gene. Deletion within the adjacent HBV enhancer element region significant reduced the activity of the X gene promoter, suggesting that the X gene promoter requires the enhancer elements (Zhou et al., 1990; Moriyama, 1997). The last region known as the epsilon-stem loop (-stem loops), the region spans nucleotides in the HBV genome and plays a key role in HBV DNA encapsidation. Transcriptional transactivator HBx protein is likely to be an important regulatory protein since its sequence is conserved among the mammalian hepadnaviridae members. X protein increases HBV transcription by trans-activating the viral enhancer l via the sequence named the E-element. HBx deregulates cell cycle checkpoints and stimulates DNA synthesis, leading to the proliferation of quiescent fibroblasts. HBx is a moderate but broad-acting transcriptional transactivator and activates a variety of cellular and viral genes, including proto-oncogenes, c-myc, c-fos, and c-jun (Spandau et al., 1988). Little is known about the exact role of HBx in tumorigenesis. Some of the reported HBV sequences have an additional in-frame initiation codon, ATG, 56 nucleotide triplets upstream of X gene s ATG epidemiological pre-x region was found (Feitelson et al., 1993). The pre-x plus

6 132 N. ABBAS AND A.R. SHAKOORI X gene, named whole-x gene, is 630 bp long and the whole-x polypeptide encoded by this gene comprises 210 amino acids. Scientist tried to identify its associated proteins in order to further reveal the biological role of whole-x protein. MUTATIONS IN THE X GENE The human HBV is the smallest known double stranded DNA virus of man that replicates through a mechanism of reverse transcription. The reverse transcriptase (RT) of HBV lacks a conventional proofreading function, therefore HBV exhibits a mutation rate more than 10-fold higher then the other known DNA viruses. The X-ORF overlaps the C-terminus of the P gene and the N- terminus of the pre C/C gene. Therefore, depending on their extension, mutations within the X-region can affect three genes at once. Mutations in the X region can involve the regulatory elements that control replication, such as the basal core promoter and Enhancer II. Because the basal core promoter encompasses nt and overlaps with the X gene in the concomitant reading frame, the A1762T plus G1764A core promoter mutations also cause changes in the X gene at xk130m and xv131i. In addition nearly all deletions or insertions in the basal core promoter results in a shift of the X gene frame and lead to the production of truncated X proteins These shortened X proteins typically lack the domain in the C terminus (amino acids ) that is required for the transactivation activity of HbxAg. X gene mutation has an important role in HbeAg expression and disease formation (Kidd- Ljunggren et al., 1997). GENERAL FEATURES OF HBx PROTEIN HBx is encoded by the smallest open reading frame of mammalian hepadnaviruses and translated from a small mrna controlled by the HBx promoter. The designation X gene/protein originally reflected its unknown function and lack of homology with known proteins. An HBx mrna has been detected in the livers of WHV-infected human liver tissue, and antibodies to HBx have been detected in some infected individuals. HBx is 154 amino acids in size, with a molecular mass of approximately 17.5 Kda. Due to a lake of results from X-ray crystallography (HBx as defied high-resolution crystallization) and nuclear magnetic resonance, little is known of

7 HEPATITIS B VIRUS: X GENE 133 HBx three-dimensional structure. Comparative analysis of HBx gene sequences from mammalian hepadnaviruses of different species revealed areas of high conservation, including presumptive helical domains located in the amino- and cabroxy-terminal regions, and a potential coiled-coil motif (Chisari, 2000). There is some evidence that disulfide bonds may link cysteine residues in the N terminus of HBx with cysteine residues in the C terminus. The HBx can be phosphorylated when expressed both in insect cells and HepG2 cells, a human hepatoblastoma cell line. Although other studies have failed to detect phosphorylation. Acetylation of HBx was observed when it was expressed in insect cells. The significance of HBx for its reported activities is currently unknown. HBx amino acids 52 to 148 are essential for its various reported activities. HBx transcriptional functions, suggesting that it may be a negative regulatory element. Higher order structures of HBx are not well established, but there is some evidence that the protein may form dimmers. HBx from both HBV and WHV display a bimodal half-life that is influenced by the intracellular location of the protein; HBx associated with the cytoskeleton and nuclear framework has a longer half-life (~3 h) HBx function is unclear but could indicate that HBx protein associated certainly detectable in the nucleus (Hu et al., 1999). In cells transfected with HBx expression vectors and during HBV and WHV infections. HBx largely in the nucleus in some cells lines. More recently, it has been reported that HBx is predominantly nuclear when expressed in cells at very low levels but becomes largely cytoplasmic as its expression level increases (Simra et al., 1998). Although these studies involved HBx could be important, considering the multiple functions of HBx during the HBV life cycle, and could influence its effects on transcriptional activation in the nucleus and viral replication in the cytoplasm (Yen, 1996). HBx IS IMPORTANT FOR THE MAMMALIAN HEPADNAVIRIDAE LIFE CYCLE Two different laboratories have demonstrated that HBx is essential for WHV infection of woodchucks. Wild type WHV genomic DNA can be directly injected in to the livers of young woodchucks, establishing a WHV infection and viremia is detectable. X is essential for virus replication in animals but dispensable for viral DNA synthesis in transfected tissue culture cells (Zoulim et al., 1994). Evidence that the gene is expressed in vivo stems from the detection of antibodies present in sera of HBV-infected humans and naturally infected animals (Persing et al., 1986; Vitvitski et al., 1990) and from protein analysis of liver samples obtained from WHV-infected woodchucks. In vitro, X exhibits a

8 134 N. ABBAS AND A.R. SHAKOORI plethora of activities. From cell culture studies, it is believed that X can activate the transcription of host genes, including the major histocompatibility complex (Hu et al., 1990) and c-myc, as well as viral genes (Colgrove et al., 1989), and one investigator has even reported that X stabilizes viral RNAs (Shimazu et al., 1998). X is not a DNA binding protein and most investigators agree that it is therefore not a typical transactivator. So, because of this its effects on transcription are thought to be indirect. Suggestions have included interacting with and altering the DNA binding of cyclic AMP-responsive element binding protein and ATF-2 (Maguire et al., 1991), activating NF- B (Twu et al., 1989), and contacting basal transcription factors). In addition to the transactivation of many promoters, activities linked to X include induction of signal transduction (Benn et al., 1994) and binding, to various degrees, to well-known protein targets such as p53 (Takada et al., 1995; Wang et al., 1994), proteasome subunits (Sirma et al., 1998; Takada et al., 1990), and UV-damaged DNA binding protein (Lee et al., 1995). Different suggestions and explanation for these and other effects of the X protein have been proposed. Whether all or any point to the role of X in the virus life cycle is not known. It is well established that X-defective virus is not able to initiate infection in vivo (Chen et al., 1993; Zoulim et al., 1994). Avian hepadnaviruses have no X gene, although DNA sequence comparisons have proposed that this gene may have been present at some time in the evolution of these viruses (Netter et al., 1997). ROLE OF X GENE IN CARCINOGENISIS Hepatitis B virus x gene and its product HBxAg possibly play an important role in carcinogenesis of hepatocellular carcinoma. HBV x gene can integrate into cellular DNA during chornic infection. HBx Ag overexpression may alter signal transduction pathways of hepatocyte. HBx Ag can bind to and inactivate negative growth regulatory suggesting its role in hepatocarcinogenesis (Zhu et al., 2004). The study with the first transgenic mouse model to express HBx in the liver reported development of liver tumors directly related to HBx expression (Kim et al., 1991). Another study showed taht for cell transformation, over expression of the viral X gene was required (Seifer et al., 1991). REFERENCES BENN, J. AND SCHNEIDER, R. J., Hepatitis B virus HBx protein activates Ras-GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade. Proc. natl. Acad. Sci. USA, 91: CHEN, H. S., KANEKO, S., GIRONES, R., ANDERSON, R. W., HORNBUCKLE, W. E.,

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