Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus.
Gene products of hepatitis C virus (HCV), a possible major causative agent of posttransfusion non-A, non-B hepatitis, are considered to be produced from a precursor polyprotein via proteolytic processing mediated by either host cell or viral proteinases. The presence of HCV serine proteinase has been proposed from analyses of amino acid sequence homology. To examine the processing mechanism of the HCV precursor polyprotein, the amino-terminal region of the putative nonstructural protein region of the HCV genome, containing the serine proteinase motif, was expressed and analyzed by using an in vitro transcription/translation system and a transient expression system in cultured cells. Two distinct proteinase activities which function in the production of a 70-kDa protein (p70) from the precursor polyprotein were detected. One of these proteinase activities, which cleaved the carboxyl (C)-terminal side of p70, required the presence of the serine proteinase motif, which is located in the amino (N)-terminal region of p70. That suggested that the predicted HCV serine proteinase was functional. The other activity, which was responsible for the cleavage of the N-terminal side of p70, required the expression of the region upstream and downstream of that cleavage site, including the p70 serine proteinase domain. From the results of pulse-chase analysis, using proteinase inhibitors coupled with a point mutation analysis, the latter activity was proposed to be a novel zinc-dependent metalloproteinase.
Virology Division, National Cancer Center Research Institute, Tokyo, Japan.
M
M
Hijikata
H
H
Mizushima
T
T
Akagi
S
S
Mori
N
N
Kakiuchi
N
N
Kato
T
T
Tanaka
K
K
Kimura
K
K
Shimotohno
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D017354:Point Mutation; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D015183:Restriction Mapping; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis*
D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; D017361:Viral Nonstructural Proteins; D010450:Endopeptidases