The conserved amino-terminal region (amino acids 1-20) of the hepatitis B virus X protein shows a transrepression function.
The X protein of hepatitis B virus or HBx is a multifunctional regulatory protein that carries the fame of a promiscuous transactivator. Although, the N-terminal 'A' region of HBx (amino acids 1-20) is the most conserved region among mammalian hepadnavirus genomes, it has been found to be dispensable for transactivation function [Proc. Natl. Acad. Sci. U.S.A. 93, 1996, 5647]. To elucidate its biological role, DNA sequence corresponding to the A region of X gene was amplified by polymerase chain reaction and cloned as a 72 base pair HBx mutant X17. In order to augment the intracellular biochemical stability of the expressed protein, the monomeric X17 was multimerized and 2-10 units long tandem repeats of the A region (X17-n) were cloned in a mammalian expression vector. Expression of the X17 constructs was confirmed by in vitro transcription and translation, as well as by RT-PCR after transfection in hepatoma cells. The function of X17 was investigated using the chloramphenicol acetyl transferase reporter constructs of viral (RSV-LTR, HIV1-LTR and HBx) and cellular gene promoters (c-Jun and epidermal growth receptor). Not only did the X17 multimers inhibit the HBx-mediated transactivation of all the reporter genes, but also their basal activities. The inhibition was dependent on the amount of X17 plasmid transfected in cells as well as on the number of repeat units present in the X17 expression vectors. Further, the X17-related inhibition of transactivation was not a cytotoxic effect. Thus, our data suggests that the N-terminal 'A' domain of HBx has a negative regulatory function.
Misra
Kamana Parashar
KP
Virology Group, International Centre for Genetic Engineering and Biotechnology, P.O. Box 10504, Aruna Asaf Ali Marg, New Delhi 110067, India.
Mukherji
Atish
A
Kumar
Vijay
V
D000818:Animals; D019677:Artificial Gene Fusion; D016466:CHO Cells; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D005814:Genes, Viral; D006515:Hepatitis B virus / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006801:Humans; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000502:physiology*; D020080:Tandem Repeat Sequences; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D054334:Viral Regulatory and Accessory Proteins
D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D015500:Chloramphenicol O-Acetyltransferase