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 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 HBx-specific 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 of the 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 C61 and C69 present in region C, caused almost 90% 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.
Kumar
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V
Virology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.
Jayasuryan
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N
Kumar
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R
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001358:Avian Sarcoma Viruses / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D017124:Conserved Sequence; D004926:Escherichia coli; D006511:Hepatitis B Antigens / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008113:Liver Neoplasms; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins
D000911:Antibodies, Monoclonal; D006511:Hepatitis B Antigens; D011994:Recombinant Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D015500:Chloramphenicol O-Acetyltransferase