Document 0403
 DOCN  M9590403
 TI    A novel method for analysis of viral proteinase activity encoded by
       hepatitis C virus in cultured cells.
 DT    9509
 AU    Hirowatari Y; Hijikata M; Shimotohno K; National Cancer Center Research
       Institute, Tokyo, Japan.
 SO    Anal Biochem. 1995 Feb 10;225(1):113-20. Unique Identifier : AIDSLINE
       GENBANK/D28816
 AB    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.
 DE    Animal  Base Sequence  Cell Line  Cercopithecus aethiops
       Chloramphenicol Acetyltransferase/ANALYSIS/BIOSYNTHESIS  DNA Primers
       Enhancer Elements (Genetics)  Fluorescent Antibody Technique  Hepatitis
       C Viruses/*ENZYMOLOGY/GENETICS  HTLV-I/GENETICS  Immunoblotting/METHODS
       Kidney  Molecular Sequence Data  Oligodeoxyribonucleotides  Peptide
       Peptidohydrolases/*BIOSYNTHESIS/GENETICS  Plasmids  Polymerase Chain
       Reaction/METHODS  Promoter Regions (Genetics)  Recombinant
       Proteins/ANALYSIS/BIOSYNTHESIS  Repetitive Sequences, Nucleic Acid
       Restriction Mapping  Support, Non-U.S. Gov't  Transfection/*METHODS
       JOURNAL ARTICLE

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