Protease Inhibitors Block Multiple Functions of the NS3/4A Protease-Helicase during the Hepatitis C Virus Life Cycle.
Hepatitis C virus (HCV) NS3 is a multifunctional protein composed of a protease domain and a helicase domain linked by a flexible linker. Protease activity is required to generate viral nonstructural (NS) proteins involved in RNA replication. Helicase activity is required for RNA replication, and genetic evidence implicates the helicase domain in virus assembly. Binding of protease inhibitors (PIs) to the protease active site blocks NS3-dependent polyprotein processing but might impact other steps of the virus life cycle. Kinetic analyses of antiviral suppression of cell culture-infectious genotype 1a strain H77S.3 were performed using assays that measure different readouts of the viral life cycle. In addition to the active-site PI telaprevir, we examined an allosteric protease-helicase inhibitor (APHI) that binds a site in the interdomain interface. By measuring nucleotide incorporation into HCV genomes, we found that telaprevir inhibits RNA synthesis as early as 12 h at high but clinically relevant concentrations. Immunoblot analyses showed that NS5B abundance was not reduced until after 12 h, suggesting that telaprevir exerts a direct effect on RNA synthesis. In contrast, the APHI could partially inhibit RNA synthesis, suggesting that the allosteric site is not always available during RNA synthesis. The APHI and active-site PI were both able to block virus assembly soon (<12 h) after drug treatment, suggesting that they rapidly engage with and block a pool of NS3 involved in assembly. In conclusion, PIs and APHIs can block NS3 functions in RNA synthesis and virus assembly, in addition to inhibiting polyprotein processing.


OBJECTIVE
The NS3/4A protease of hepatitis C virus (HCV) is an important antiviral target. Currently, three PIs have been approved for therapy of chronic hepatitis C, and several others are in development. NS3-dependent cleavage of the HCV polyprotein is required to generate the mature nonstructural proteins that form the viral replicase. Inhibition of protease activity can block RNA replication by preventing expression of mature replicase components. Like many viral proteins, NS3 is multifunctional, but how PIs affect stages of the HCV life cycle beyond polyprotein processing has not been well studied. Using cell-based assays, we show here that PIs can directly inhibit viral RNA synthesis and also block a late stage in virus assembly/maturation at clinically relevant concentrations.
Division of Infectious Diseases, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Lineberger Comprehensive Cancer Center, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA mcgivern@med.unc.edu.
David R
DR
McGivern
Division of Infectious Diseases, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Lineberger Comprehensive Cancer Center, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Takahiro
T
Masaki
Division of Infectious Diseases, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Lineberger Comprehensive Cancer Center, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
William
W
Lovell
Astex Pharmaceuticals, Cambridge, United Kingdom.
Chris
C
Hamlett
Astex Pharmaceuticals, Cambridge, United Kingdom.
Susanne
S
Saalau-Bethell
Astex Pharmaceuticals, Cambridge, United Kingdom.
Brent
B
Graham
D000998:Antiviral Agents / Q000494:pharmacology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000502:physiology*; D002460:Cell Line; D016174:Hepacivirus / Q000187:drug effects* / Q000502:physiology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D009842:Oligopeptides / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012367:RNA, Viral / Q000096:biosynthesis; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000502:physiology*; D019065:Virus Assembly / Q000187:drug effects* / Q000502:physiology*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000998:Antiviral Agents; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009842:Oligopeptides; D011480:Protease Inhibitors; 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; C455041:NS4A cofactor peptide, Hepatitis C virus; C486464:telaprevir