Kinetic analyses reveal potent and early blockade of hepatitis C virus assembly by NS5A inhibitors.
OBJECTIVE
All-oral regimens combining different classes of direct-acting antivirals (DAA) are highly effective for treatment of patients with chronic hepatitis C. NS5A inhibitors will likely form a component of future interferon-sparing treatment regimens. However, despite their potential, the detailed mechanism of action of NS5A inhibitors is unclear. To study their mechanisms, we compared their kinetics of antiviral suppression with those of other classes of DAA, using the hepatitis C virus genotype 1a cell culture-infectious virus H77S.3.


METHODS
We performed detailed kinetic analyses of specific steps in the hepatitis C virus life cycle using cell cultures incubated with protease inhibitors, polymerase inhibitors, or NS5A inhibitors. Assays were designed to measure active viral RNA synthesis and steady-state RNA abundance, polyprotein synthesis, virion assembly, and infectious virus production.


RESULTS
Despite their high potency, NS5A inhibitors were slow to inhibit viral RNA synthesis compared with protease or polymerase inhibitors. By 24 hours after addition of an NS5A inhibitor, polyprotein synthesis was reduced <50%, even at micromolar concentrations. In contrast, inhibition of virus release by NS5A inhibitors was potent and rapid, with onset of inhibition as early as 2 hours. Cells incubated with NS5A inhibitors were rapidly depleted of intracellular infectious virus and RNA-containing hepatitis C virus particles, indicating a block in virus assembly.


CONCLUSIONS
DAAs that target NS5A rapidly inhibit intracellular assembly of genotype 1a virions. They also inhibit formation of functional replicase complexes, but have no activity against preformed replicase, thereby resulting in slow shut-off of viral RNA synthesis.
Departments of Medicine and Microbiology and Immunology and the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. Electronic address: mcgivern@med.unc.edu.
David R
DR
McGivern
Departments of Medicine and Microbiology and Immunology and the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
Takahiro
T
Masaki
Departments of Medicine and Microbiology and Immunology and the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
Sara
S
Williford
Merck Research Laboratory, Kenilworth, New Jersey.
Paul
P
Ingravallo
Departments of Medicine and Microbiology and Immunology and the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
Zongdi
Z
Feng
Merck Research Laboratory, Kenilworth, New Jersey.
Frederick
F
Lahser
Merck Research Laboratory, Kenilworth, New Jersey.
Ernest
E
Asante-Appiah
Fondazione INGM, Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi", 20122 Milan, Italy.
Petra
P
Neddermann
Fondazione INGM, Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi", 20122 Milan, Italy.
Raffaele
R
De Francesco
Merck Research Laboratory, Kenilworth, New Jersey.
Anita Y
AY
Howe
Departments of Medicine and Microbiology and Immunology and the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. Electronic address: smlemon@med.unc.edu.
Stanley M
SM
Lemon
D000998:Antiviral Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D024882:Drug Resistance, Viral; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005838:Genotype; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D006801:Humans; D007700:Kinetics; D009154:Mutation; D011480:Protease Inhibitors / Q000494:pharmacology; D012367:RNA, Viral / Q000096:biosynthesis; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly / Q000187:drug effects*; D014779:Virus Replication / Q000187:drug effects; D012324:RNA-Dependent RNA Polymerase
D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D011480:Protease Inhibitors; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
Antiviral Agent; RNA-Dependent RNA Polymerase; Replication; Therapy