Quantitative analysis of the hepatitis C virus replication complex.
The hepatitis C virus (HCV) encodes a large polyprotein; therefore, all viral proteins are produced in equimolar amounts regardless of their function. The aim of our study was to determine the ratio of nonstructural proteins to RNA that is required for HCV RNA replication. We analyzed Huh-7 cells harboring full-length HCV genomes or subgenomic replicons and found in all cases a >1,000-fold excess of HCV proteins over positive- and negative-strand RNA. To examine whether all nonstructural protein copies are involved in RNA synthesis, we isolated active HCV replication complexes from replicon cells and examined them for their content of viral RNA and proteins before and after treatment with protease and/or nuclease. In vitro replicase activity, as well as almost the entire negative- and positive-strand RNA, was resistant to nuclease treatment, whereas <5% of the nonstructural proteins were protected from protease digest but accounted for the full in vitro replicase activity. In consequence, only a minor fraction of the HCV nonstructural proteins was actively involved in RNA synthesis at a given time point but, due to the high amounts present in replicon cells, still representing a huge excess compared to the viral RNA. Based on the comparison of nuclease-resistant viral RNA to protease-resistant viral proteins, we estimate that an active HCV replicase complex consists of one negative-strand RNA, two to ten positive-strand RNAs, and several hundred nonstructural protein copies, which might be required as structural components of the vesicular compartments that are the site of HCV replication.
Quinkert
Doris
D
Department Molecular Virology, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany.
Bartenschlager
Ralf
R
Lohmann
Volker
V
D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D016174:Hepacivirus / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D012367:RNA, Viral / Q000032:analysis / Q000096:biosynthesis*; D017361:Viral Nonstructural Proteins / Q000032:analysis / Q000096:biosynthesis*; D014779:Virus Replication
D012367:RNA, Viral; D017361:Viral Nonstructural Proteins