Construction of hepatitis C-SIN virus recombinants with replicative dependency on hepatitis C virus serine protease activity.
An in vivo assay system was developed for the serine protease of hepatitis C virus (HCV) using the sindbis (SIN) viral replication system in which HCV serine protease activity is essential for the replication of the HCV-SIN chimeric virus. Two chimeric viral cDNA clones were constructed by inserting the NS3/4A region and NS3/4A region with the putative helicase deleted, into the N-terminal region of SIN core protein. The constructs were named Tpro CT and Tpro T, respectively. BHK-21 cells transfected with the in vitro transcribed RNAs from Tpro CT and Tpro T showed specific cytopathic morphology and produced chimeric viruses, Vpro CT and Vpro T. In contrast, in vitro transcribed RNAs from Tpro CTI and Tpro TI, in which serine of catalytic triad of HCV protease was changed to alanine, were not infectious. When the chimeric viruses were passaged in BHK-21 cells at about 0.1 multiplicity of infection (MOI), Vpro T, but not Vpro CT, stably expressed HCV protease for up to five passages. Surprisingly, the cell culture media of BHK-21 cells infected with Vpro T, compared to wild-type sindbis virus, showed rapid pH changes by more than 0.8 pH degree at 72 h post-infection. HCV-SIN hybrid viruses could be used in screening the HCV protease-inhibitor in cell culture systems.
Cho
Y G
YG
Department of Life Science, School of Environmental Engineering, Pohang University of Science and Technology, South Korea.
Moon
H S
HS
Sung
Y C
YC
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D017077:Culture Media, Conditioned / Q000737:chemistry; D003545:Cysteine / Q000032:analysis; D004274:DNA, Recombinant / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D005822:Genetic Vectors; D016679:Genome, Viral; D016174:Hepacivirus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008715:Methionine / Q000032:analysis; D011233:Precipitin Tests; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000737:chemistry / Q000494:pharmacology*; D012845:Sindbis Virus / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D013462:Sulfur Radioisotopes / Q000032:analysis; D014162:Transfection; D014779:Virus Replication / Q000187:drug effects* / Q000235:genetics
D017077:Culture Media, Conditioned; D004274:DNA, Recombinant; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D013462:Sulfur Radioisotopes; D008715:Methionine; D012697:Serine Endopeptidases; D003545:Cysteine