Hepatitis C virus non-structural proteins in the probable membranous compartment function in viral genome replication.
The molecular mechanism of hepatitis C virus(HCV) RNA replication is still unknown. Recently, a cell culture system in which the HCV subgenomic replicon is efficiently replicated and maintained for a long period in Huh-7 cells has been established. Taking advantage of this replicon system, we detected the activity to synthesize the subgenomic RNA in the digitonin-permeabilized replicon cells. To elucidate how and where this viral RNA replicates in the cells, we monitored the activity for HCV RNA synthesis in the permeabilized replicon cells under several conditions. We obtained results suggesting that HCV replication complexes functioning to synthesize the replicon RNA are protected from access of nuclease and proteinase by possible cellular lipid membranes. We also found that a large part of the replicon RNA, including newly synthesized RNA, was present in such a membranous structure but a large part of each NS protein was not. A small part of each NS protein that was resistant to the proteinase action was shown to contribute sufficiently to the synthesis of HCV subgenomic RNA in the permeabilized replicon cells. These results suggested that a major subcellular site of HCV genome replication is probably compartmentalized by lipid membranes and that only a part of each NS protein forms the active replication complex in the replicon cells.
Miyanari
Yusuke
Y
Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.
Hijikata
Makoto
M
Yamaji
Masashi
M
Hosaka
Masahiro
M
Takahashi
Hitoshi
H
Shimotohno
Kunitada
K
D015152:Blotting, Northern; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D004072:Digitonin / Q000494:pharmacology; D019286:Endopeptidase K / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D016679:Genome, Viral; D016174:Hepacivirus / Q000378:metabolism*; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D050356:Lipid Metabolism; D008856:Microscopy, Fluorescence; D009693:Nucleic Acid Hybridization; D010957:Plasmids / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism; D014779:Virus Replication
D007202:Indicators and Reagents; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D019286:Endopeptidase K; D004072:Digitonin