NS3 Peptide, a novel potent hepatitis C virus NS3 helicase inhibitor: its mechanism of action and antiviral activity in the replicon system.
Hepatitis C virus (HCV) chronic infections represent one of the major and still unresolved health problems because of low efficiency and high cost of current therapy. Therefore, our studies centered on a viral protein, the NS3 helicase, whose activity is indispensable for replication of the viral RNA, and on its peptide inhibitor that corresponds to a highly conserved arginine-rich sequence of domain 2 of the helicase. The NS3 peptide (p14) was expressed in bacteria. Its 50% inhibitory activity in a fluorometric helicase assay corresponded to 725 nM, while the ATPase activity of NS3 was not affected. Nuclear magnetic resonance (NMR) studies of peptide-protein interactions using the relaxation filtering technique revealed that p14 binds directly to the full-length helicase and its separately expressed domain 1 but not to domain 2. Changes in the NMR chemical shift of backbone amide nuclei ((1)H and (15)N) of domain 1 or p14, measured during complex formation, were used to identify the principal amino acids of both domain 1 and the peptide engaged in their interaction. In the proposed interplay model, p14 contacts the clefts between domains 1 and 2, as well as between domains 1 and 3, preventing substrate binding. This interaction is strongly supported by cross-linking experiments, as well as by kinetic studies performed using a fluorometric assay. The antiviral activity of p14 was tested in a subgenomic HCV replicon assay that showed that the peptide at micromolar concentrations can reduce HCV RNA replication.
Institute of Biochemistry and Biophysics PAS, ul. Pawinskiego 5a, 02-106 Warsaw, Poland.
Agnieszka
A
Gozdek
Igor
I
Zhukov
Agnieszka
A
Polkowska
Jaroslaw
J
Poznanski
Anna
A
Stankiewicz-Drogon
Jerzy M
JM
Pawlowicz
Wlodzimierz
W
Zagórski-Ostoja
Peter
P
Borowski
Anna M
AM
Boguszewska-Chachulska
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D020365:RNA Helicases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D012367:RNA, Viral / Q000378:metabolism; D012093:Replicon / Q000187:drug effects*; D017361:Viral Nonstructural Proteins / Q000737:chemistry*; D014779:Virus Replication / Q000187:drug effects; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D010455:Peptides; D020365:RNA Helicases; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases