Analysis of the X gene promoter of woodchuck hepatitis virus.
During the course of woodchuck hepatitis virus (WHV) replication three virus-specific mRNA transcripts that encode four essential proteins are produced. The transcripts are 3.6, 2.3, and 0.7 kb in size. The 3.6-kb transcript serves as the replicative intermediate as well as the template for translation of the nucleocapsid and polymerase proteins. The 2.3-kb mRNA serves as the template for translation of the virus envelope proteins. Both the 3.6- and 2.3-kb transcripts are polyadenylated and are readily found in the cytoplasm of infected hepatocytes. However, the 0.7-kb transcript, specific for the X gene, accumulates in the nucleus of infected cells and is polyadenylated poorly in hepatocytes. Thus, while it is likely that the 0.7-kb transcript is the template for translation of the X protein, it is possible that it also has a function at the RNA level to regulate virus replication or gene expression. In order to characterize the WHV X promoter we cloned the region of the WHV8 genome encompassing the viral enhancer through the amino terminus of the X gene into the vector pSV0CAT. We transfected Huh-7 and WLC-3 cells with the WHV X promoter construct, along with a plasmid encoding human growth hormone to control for transfection efficiency, and assayed for the presence of chloramphenicol acetyl transferase activity. We found that the WHV X promoter was about one-half as active as the well-studied simian virus 40 early or Rous sarcoma virus promoters. Next, we made a series of 5' and 3' deletion mutants and mapped the WHV X promoter to a 21-nucleotide domain (1482-GGGGAAGCTGACGTCCTTTCC-1502) which is approximately 100 bp downstream of the corresponding promoter in hepatitis B virus. Further analysis, using oligonucleotide-directed mutagenesis, demonstrated that the essential nucleotides comprising the WHV X promoter are located in a 10-nucleotide domain near the initiation codon of the X gene. Mutation of either nucleotide T at position 1490 or G at position 1491 within this domain was sufficient to reduce the level of promoter activity by 100-fold. Thus, we have defined the important nucleotides within the promoter of the WHV X transcript which is a first step in understanding the role of this transcript in WHV replication and gene expression.
Sugata
F
F
Hepatitis Virus Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Chen
H S
HS
Kaneko
S
S
Purcell
R H
RH
Miller
R H
RH
D000818:Animals; D001483:Base Sequence; D017919:Hepatitis B Virus, Woodchuck / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion; D015534:Trans-Activators / Q000235:genetics*; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D015534:Trans-Activators; D014764:Viral Proteins; C090237:protein X, Woodchuck hepatitis B virus