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PMID: 8643631 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't

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

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 93 ·No. 11 ·1996-05-28 ·Pages 5647-52

Kumar V, Jayasuryan N, Kumar R

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 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.

MeSH Terms
Amino Acid Sequence Animals Antibodies, Monoclonal Avian Sarcoma Viruses/genetics Base Sequence Blotting, Western Carcinoma, Hepatocellular Cell Line Chloramphenicol O-Acetyltransferase/biosynthesis Conserved Sequence Escherichia coli Hepatitis B Antigens/metabolism Hepatitis B virus/genetics,metabolism Humans Liver Neoplasms Mice Mice, Inbred BALB C/immunology Molecular Sequence Data Mutagenesis, Site-Directed Polymerase Chain Reaction Recombinant Proteins/isolation & purification,metabolism Repetitive Sequences, Nucleic Acid Sequence Deletion Sequence Homology, Amino Acid Trans-Activators/genetics,isolation & purification,metabolism Transcriptional Activation Transfection Viral Regulatory and Accessory Proteins
Chemicals
Antibodies, Monoclonal Hepatitis B Antigens Recombinant Proteins Trans-Activators Viral Regulatory and Accessory Proteins hepatitis B virus X protein Chloramphenicol O-Acetyltransferase
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Kumar V
Virology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.
Jayasuryan N
Kumar R
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.93.11.5647
Published
1996-05-28
Pages
5647-52
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC39302
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.93.11.5647 PMC Full Text View journal details
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