title: Fast hepatitis C virus RNA elimination and NS5A redistribution by NS5A inhibitors studied by a multiplex assay approach.
abstract: While earlier therapeutic strategies for the treatment of hepatitis C virus (HCV) infection relied exclusively on interferon (IFN) and ribavirin (RBV), four direct-acting antiviral agents (DAAs) have now been approved, aiming for an interferon-free strategy with a short treatment duration and fewer side effects. To facilitate studies on the mechanism of action (MOA) and efficacy of DAAs, we established a multiplex assay approach, which employs flow cytometry, a Gaussia luciferase reporter system, Western blot analysis, reverse transcription-quantitative PCR (RT-qPCR), a limited dilution assay (50% tissue culture infectious dose [TCID50]), and an image profiling assay that follows the NS5A redistribution in response to drug treatment. We used this approach to compare the relative potency of various DAAs and the kinetics of their antiviral effects as a potential preclinical measure of their potential clinical utility. We evaluated the NS5A inhibitors ledipasvir (LDV) and daclatasvir (DCV), the NS3/4A inhibitor danoprevir (DNV), and the NS5B inhibitor sofosbuvir (SOF). In terms of kinetics, our data demonstrate that the NS5A inhibitor LDV, followed closely by DCV, has the fastest effect on suppression of viral proteins and RNA and on redistribution of NS5A. In terms of MOA, LDV has a more pronounced effect than DCV on the viral replication, assembly, and infectivity of released virus. Our approach can be used to facilitate the study of the biological processes involved in HCV replication and help identify optimal drug combinations.
authors.affiliation: Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, Missouri, USA Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, Missouri, USA.
authors.forename: Dandan
authors.initials: D
authors.lastname: Liu
authors.affiliation: Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, Missouri, USA Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, Missouri, USA.
authors.forename: Juan
authors.initials: J
authors.lastname: Ji
authors.affiliation: Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, Missouri, USA Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, Missouri, USA.
authors.forename: Tanya P
authors.initials: TP
authors.lastname: Ndongwe
authors.affiliation: The Rockefeller University, Laboratory of Virology and Infectious Disease, New York, New York, USA.
authors.forename: Eleftherios
authors.initials: E
authors.lastname: Michailidis
authors.affiliation: The Rockefeller University, Laboratory of Virology and Infectious Disease, New York, New York, USA.
authors.forename: Charles M
authors.initials: CM
authors.lastname: Rice
authors.affiliation: Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Ralston
authors.affiliation: Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, Missouri, USA Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, Missouri, USA Department of Biochemistry, University of Missouri, Columbia, Missouri, USA sarafianoss@missouri.edu.
authors.forename: Stefan G
authors.initials: SG
authors.lastname: Sarafianos
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002219:Carbamates; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology; D011759:Pyrrolidines; D012367:RNA, Viral / Q000235:genetics*; D012254:Ribavirin / Q000494:pharmacology; D000069474:Sofosbuvir / Q000494:pharmacology; D014633:Valine / Q000031:analogs & derivatives; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D002219:Carbamates; D007093:Imidazoles; D011759:Pyrrolidines; D012367:RNA, Viral; D012254:Ribavirin; D000069474:Sofosbuvir; D014633:Valine; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C549273:daclatasvir
references.citation: N Engl J Med. 2014 Apr 17;370(16):1483-93
references.pmid: 24725238
references.citation: J Virol. 2011 Jul;85(14):7460-4
references.pmid: 21593166
references.citation: J Hepatol. 2011 Jul;55(1):69-75
references.pmid: 21145856
references.citation: Gastroenterology. 2014 Aug;147(2):453-62.e7
references.pmid: 24768676
references.citation: J Virol. 2008 Aug;82(16):7964-76
references.pmid: 18524832
references.citation: Proc Natl Acad Sci U S A. 2006 May 9;103(19):7408-13
references.pmid: 16651538
references.citation: Nat Rev Microbiol. 2013 Jul;11(7):482-96
references.pmid: 23748342
references.citation: Gastroenterology. 2014 Nov;147(5):1094-105.e25
references.pmid: 25046163
references.citation: Virus Res. 2007 Aug;127(2):195-207
references.pmid: 17428568
references.citation: Methods Mol Biol. 2009;510:33-53
references.pmid: 19009252
references.citation: Gut. 2007 Aug;56(8):1111-6
references.pmid: 17363475
references.citation: Hepatology. 2011 Oct;54(4):1433-44
references.pmid: 21898493
references.citation: Genome Biol. 2006;7(10):R100
references.pmid: 17076895
references.citation: J Biomol Screen. 2010 Aug;15(7):840-6
references.pmid: 20516293
references.citation: PLoS Pathog. 2011;7(2):e1001278
references.pmid: 21347350
references.citation: Virus Res. 2012 Dec;170(1-2):1-14
references.pmid: 23009750
references.citation: Clin Infect Dis. 2012 Jul;55 Suppl 1:S10-5
references.pmid: 22715208
references.citation: N Engl J Med. 2014 May 15;370(20):1889-98
references.pmid: 24725239
references.citation: J Hepatol. 2005 Sep;43(3):425-33
references.pmid: 15990196
references.citation: Nat Methods. 2012 Jul;9(7):676-82
references.pmid: 22743772
references.citation: Science. 2005 Jul 22;309(5734):623-6
references.pmid: 15947137
references.citation: Proc Natl Acad Sci U S A. 2005 Jun 28;102(26):9294-9
references.pmid: 15939869
references.citation: Curr Top Microbiol Immunol. 2013;369:1-15
references.pmid: 23463195
references.citation: Swiss Med Wkly. 2012;142:w13516
references.pmid: 22367957
references.citation: Acta Gastroenterol Belg. 2012 Jun;75(2):245-59
references.pmid: 22870791
references.citation: J Hepatol. 2011 Jun;54(6):1130-6
references.pmid: 21145848
references.citation: Biotechniques. 2007 Jan;42(1):71-5
references.pmid: 17269487
references.citation: J Virol. 2008 Feb;82(3):1073-83
references.pmid: 18032500
references.citation: Curr Opin Investig Drugs. 2010 Aug;11(8):951-63
references.pmid: 20721837
references.citation: Hepat Med. 2014 Apr 29;6:25-33
references.pmid: 24822024
references.citation: J Virol. 2013 Dec;87(24):13297-306
references.pmid: 24089562
references.citation: J Virol. 2009 Sep;83(17):8379-95
references.pmid: 19515772
references.citation: Nature. 2005 Aug 18;436(7053):933-8
references.pmid: 16107832
references.citation: Curr Opin Microbiol. 2002 Aug;5(4):419-27
references.pmid: 12160863
references.citation: Proc Natl Acad Sci U S A. 2013 Mar 5;110(10):3991-6
references.pmid: 23431163
references.citation: Hepatology. 2008 Jan;47(1):35-42
references.pmid: 17975791
references.citation: Annu Rev Med. 2012;63:373-87
references.pmid: 21942423
references.citation: Bioinformatics. 2011 Apr 15;27(8):1179-80
references.pmid: 21349861
references.citation: Therap Adv Gastroenterol. 2014 May;7(3):131-40
references.pmid: 24790644
references.citation: Hepatology. 2008 Dec;48(6):1843-50
references.pmid: 19003912
references.citation: Nature. 2010 May 6;465(7294):96-100
references.pmid: 20410884
references.citation: Hepatology. 2011 Dec;54(6):1924-35
references.pmid: 21809362
references.citation: J Gen Virol. 2004 Sep;85(Pt 9):2485-502
references.pmid: 15302943
references.citation: Virology. 2011 May 25;414(1):10-8
references.pmid: 21513964
references.citation: J Hepatol. 2013 Aug;59(2):375-82
references.pmid: 23567084
references.citation: J Virol. 2011 Jul;85(13):6353-68
references.pmid: 21507963