A truncated mutant (residues 58-140) of the hepatitis B virus X protein retains transactivation function.

Abstract

The hepatitis B virus X protein (HBx) sequence (154 aa) has been divided into six regions (A-F) based on its sequence homology with X proteins of other mammalian hepadnaviruses. Regions A, C, and E are more conserved and include all the four conserved cysteines (C7, C61, C69, and C137).To localize the regions of HBx important for transactivation, a panel of 10 deletion mutants (X5-X14) and 4 single point mutants (X1-X4), each corresponding to a conserved cysteine residue, was constructed by site-directed mutagenesis. A HBxspecific monoclonal antibody was developed and used to confirm the expression of mutants by Western blot. Transactivation property of the HBx mutants was studied on Rous sarcoma virus-long terminal repeat (RSV-LTR) in transient transfection assays. We observed that deletion of the most conserved region A or substitution of the N-terminal cysteine (C7) had no effect on transactivation. Deletion ofthe nonconserved regions B or F also had no deleterious effects. Deletions of regions C and D resulted in a significant loss of function. Substitution of both C6' and C69 present in region C, caused almost 90%0o loss of activity that could be partially overcome by transfecting more expression plasmid. The fully conserved 9 amino acid segment (residues 132 to 140)within region E including C137 appeared to be crucial for its activity. Finally, a truncated mutant X15 incorporating only regions C to E (amino acids 58-140) was able to stimulate the RSV-LTR quite efficiently, suggesting a crucial role played by this domain in transactivation function.Hepatitis B virus (HBV) is the infective agent for the widespread liver disease in humans known as hepatitis B. Chronic infection has been associated with a high risk for development of hepatocellular carcinoma (1, 2). Similar viruses are also found in several animal species such as woodchucks, ground squirrels, and ducks, and together they constitute the family Hepadnaviridae. The small 3.2-kb DNA genome of HBV has at least four open reading frames called S, C, P, and X. During the natural course of HBV infection, the X gene expresses a polypeptide (HBx) of 154 residues that is implicated in HBVmediated hepatocellular carcinoma (3, 4).HBx is a pleiotropic transactivator because it can stimulate the cis-elements of not only the HBV genome (5, 6) but also of a wide range of other viral promoters such as simian virus 40 early (5, 7, 8) and herpes simplex virus-tk (7, 8), and long terminal repeats (LTRs) of human immunodeficiency virus type 1 (9, 10), human immunodeficiency virus type 2 (11), human T-lymphotropic virus type 1 (7), mouse mammary tumor virus (5, 7), and Rous sarcoma virus (RSV) (7,8). HBx can also upregulate the expression of some protooncogenes like c-myc (12) and c-fos/c-jun (13)(14)(15) MATERIALS AND METHODSHBx Expression Vector and HBx Mutants. The HBx gene was amplified as a 481-bp DNA fragment by PCR using the full-length HBV template (adw subtype) (38) and the following oligonucleotide primers: forward, 5'-CGGAATTCATGGCT-GCTAGGCTGT-...

UVDDB p127-binding modulates activities and intracellular distribution of Hepatitis B virus X protein

“…Interaction with UVDDB represents an upstream step in X mode of action and modulates nuclear targeting of the viral protein Importantly, the minimal domain necessary for HBxmediated transactivation that was previously mapped to region 58-140 (Kumar et al, 1996) extends downstream of the UVDDB-binding domain, which ends around residue 100 in both HBx and WHx ( (Sitterlin et al, 1997) and this study). Therefore, in addition to interaction with UVDDB, X carries at least one other activity.…”

c-Fos Is a Mediator of the c-Myc-induced Apoptotic Signaling in Serum-deprived Hepatoma Cells via the p38 Mitogen-activated Protein Kinase Pathway

“…Expression Vectors and Reporter Gene Constructs-We have described previously (38) the construction of the HBx expression vector * This work was supported by core grants from the International Centre for Genetic Engineering and Biotechnology, New Delhi. The costs of publication of this article were defrayed in part by the payment of page charges.…”

HBX causes cyclin D1 overexpression and development of breast cancer in transgenic animals that are heterozygous for p53

“…Furthermore, related studies demonstrated that truncated HBX molecules, lacking the p53-binding region, still transform tissue culture cells (Kumar et al, 1996;Gottlob et al, 1998a).…”