Expression and processing of putative nonstructural proteins of hepatitis C virus in insect cells using baculovirus vector.
Processing of the putative nonstructural (NS) proteins, p70(NS3), p4(NS4A), p27(NS4B), p58/56(NS5A), and p66(NS5B), of Japanese type hepatitis C virus (HCV) in insect cells was analyzed by using a baculovirus expression system. Products processed by the HCV serine proteinase (Cpro-2) were essentially identical to those found in mammalian cultured cells transiently producing the NS region of the HCV precursor polyprotein. A series of internal and carboxy (C)-terminal deletion experiments coupled with epitope scanning analysis showed that efficient cleavage at the Cpro-2-dependent processing sites, except at the p4(NS4A)/p27(NS4B) site, is not significantly influenced by those mutations. Efficient cleavage at p4(NS4A)/p27(NS4B) required about 40% of the NS5A N-terminal region. Estimation of the processing sites by determination of the N-terminal amino acid sequences of the processed products revealed that all the Cpro-2-dependent cleavages occurred at essentially identical sites to those reported for another HCV genotype, suggesting that Cpro-2 is a possible target for the development of a strain-independent anti-HCV agent.
Hirowatari
Y
Y
Virology Division, National Cancer Center Research Institute, Tokyo, Japan.
Hijikata
M
M
Tanji
Y
Y
Shimotohno
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D016174:Hepacivirus / Q000235:genetics*; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
D010450:Endopeptidases; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C084816:proteinase Cpro-2, hepatitis C virus