A novel method for analysis of viral proteinase activity encoded by hepatitis C virus in cultured cells.
We developed a novel method for analysis of hepatitis C viral proteinase activity in cultured cells, in which the proteinase activity was measured as the enhancement of reporter gene expression. In this system, plasmids encoding a reporter gene, the enzyme gene, and the substrate gene were simultaneously transfected into COS-1 cells. The reporter plasmid contains chloramphenicol acetyltransferase (CAT) gene downstream of an enhancer/promoter sequence derived from the human T-cell leukemia virus type-1 (HTLV-I) long-terminal repeat (LTR). The substrate expression plasmid was a triple chimera; HCV nonstructural protein 2 (NS2) and the Tax1 protein of HTLV-I sandwiched the substrate polypeptide, which was inserted upstream of Tax1. This method assumes that since the HCV NS2 appears to be located in the lipid bilayer of endoplasmic reticulum (ER) membranes, the Tax1 of the chimeric substrate was trapped on the surface of the ER in the absence of HCV proteinase activity. After release from the chimera by HCV proteinase-dependent cleavage, Tax1 could transactivate the expression of the CAT gene through the enhancer sequence of HTLV-I LTR. This system should enable us to simply and safely screen the potential antiviral activity of proteinase inhibitors in vivo, although this system may be limited to proteinase inhibitors that are permeable to the plasma membrane.
Hirowatari
Y
Y
National Cancer Center Research Institute, Tokyo, Japan.
Hijikata
M
M
Shimotohno
K
K
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000096:biosynthesis; D002522:Chlorocebus aethiops; D017931:DNA Primers; D010450:Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D004742:Enhancer Elements, Genetic; D005455:Fluorescent Antibody Technique; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D015368:Human T-lymphotropic virus 1 / Q000235:genetics; D015151:Immunoblotting / Q000379:methods; D007668:Kidney; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D014162:Transfection / Q000379:methods*
D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D015500:Chloramphenicol O-Acetyltransferase; D010450:Endopeptidases