A TRUNCATED MUTANT (RESIDUES-58-140) OF THE HEPATITIS-B VIRUS-X PROTEIN RETAINS TRANSACTIVATION FUNCTION

The hepatitis B virus X protein (HBx) sequence (154 aa) has been divid ed into six regions (A-F) based on its sequence homology with X protei ns of other mammalian hepadnaviruses. Regions A, C, and E are more con served and include all the four conserved cysteines (C-7, C-61, C-69, and C-137) To localize the regions of HBx important for transactivatio n, a panel of 10 deletion mutants (X5-X14) and 4 single point mutants (X1-X4), each corresponding to a conserved cysteine residue, was const ructed by site-directed mutagenesis. A HBx-specific monoclonal antibod y was developed and used to confirm the expression of mutants by Weste rn blot. Transactivation property of the HBx mutants was studied on Ro us sarcoma virus-long terminal repeat (RSV-LTR) in transient transfect ion assays. We observed that deletion of the most conserved region A o r substitution of the N-terminal cysteine (C-7) had no effect on trans activation. Deletion of the nonconserved regions B or F also had no de leterious effects. Deletions of regions C and D resulted in a signific ant loss of function. Substitution of both C-61 and C-69 present in re gion C, caused almost 90% loss of activity that could be partially ove rcome by transfecting more expression plasmid. The fully conserved 9 a mino acid segment (residues 132 to 140) within region E including C-13 7 appeared to be crucial for its activity. Finally, a truncated mutant X15 incorporating only regions C to E (amino acids 58-140) was able t o stimulate the RSV-LTR quite efficiently, suggesting a crucial role p layed by this domain in transactivation function.