title: Hepatitis C virus helicase/NTPase: an efficient expression system and new inhibitors.
abstract: A method has been developed for obtaining a full-length protein NS3 of hepatitis C virus with the yield of 6.5 mg/liter of cell culture, and conditions for measuring its NTPase and helicase activities have been optimized. The helicase reaction can proceed in two modes depending on the enzyme and substrate concentration ratio: it can be non-catalytic in the case of enzyme excess and catalytic in the case of tenfold substrate excess. In the latter case, helicase activity is coupled with NTPase and is stimulated by ATP. A number of NTP and inorganic pyrophosphate analogs were studied as substrates and/or inhibitors of NS3 NTPase activity, and it was found that the structure of nucleic base and ribose fragment of NTP molecule has a slight effect on its inhibitory (substrate) properties. Among the nucleotide derivatives, the most efficient inhibitor of NTPase activity is 2 -deoxythymidine 5 -phosphoryl-beta,gamma-hypophosphate, and among pyrophosphate analogs imidodiphosphate exhibited maximal inhibitory activity. These compounds were studied as inhibitors of the helicase reaction, and it was shown that imidodiphosphate efficiently inhibited the ATP-dependent helicase reaction and had almost no effect on the ATP-independent duplex unwinding. However, the inhibitory effect of 2 -deoxythymidine 5 -phosphoryl-beta,gamma-hypophosphate was insignificant in both cases, which is due to the possibility of helicase activation by this ATP analog.
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow 119991, Russia.
authors.forename: A V
authors.initials: AV
authors.lastname: Mukovnya
authors.forename: V L
authors.initials: VL
authors.lastname: Tunitskaya
authors.forename: A L
authors.initials: AL
authors.lastname: Khandazhinskaya
authors.forename: N A
authors.initials: NA
authors.lastname: Golubeva
authors.forename: N F
authors.initials: NF
authors.lastname: Zakirova
authors.forename: A V
authors.initials: AV
authors.lastname: Ivanov
authors.forename: M K
authors.initials: MK
authors.lastname: Kukhanova
authors.forename: S N
authors.initials: SN
authors.lastname: Kochetkov
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D004526:Efficiency; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D008274:Magnesium / Q000494:pharmacology; D008954:Models, Biological; D043583:Nucleoside-Triphosphatase / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000255:Adenosine Triphosphate; D004791:Enzyme Inhibitors; D008274:Magnesium; D043583:Nucleoside-Triphosphatase; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases