title: Sensitivity of a ribavirin resistant mutant of hepatitis C virus to other antiviral drugs.
abstract: BACKGROUND
  While ribavirin mono-therapy regimens have minimal effect on patients with chronic hepatitis C virus (HCV) infections, they can be efficacious when combined with interferon. Clinical studies show that interferon-free combination therapies containing ribavirin are also efficacious, suggesting that an interferon-free therapy could be adopted in the near future. However, generation of drug resistant mutants and cross resistance to other drugs could impair the efficacy of the treatment. Therefore, understanding the mechanism of HCV resistance to ribavirin and cross resistance to other antiviral drugs could be of major importance.
  
  
  METHODS
  We tested the ability of a J6/JFH1 derived HCV ribavirin resistant mutant to grow in tissue cultured Huh7D cells in the presence of the mutagen 5-Fluorouracil and the nucleoside analog 2'-C-Methylcytidine. Virus replication was assessed by detecting HCV antigens by immunofluorescence and by titrating virus present in the supernatants. Recovered viruses were amplified by RT-PCR and sequenced.
  
  
  RESULTS
  The sensitivity of HCV-RR relative to parental J6/JFH1 to the tested drugs varied. HCV-RR was more resistant than J6/JFH1 to 5-Fluorouracil but was not more resistant than J6/JFH1 to 2'-C-Methylcytidine. Growth of HCV-RR in 5-Fluorouracil allowed the selection of an HCV-RR derived mutant resistant to 5-Fluorouracil (HCV-5FU). HCV-5FU grows to moderate levels in the presence of high concentrations of 5-Fluorouracil and to parental levels in the absence of the drug. Sequence of its genome shows that HCV-5FU accumulated multiple synonymous and non-synonymous mutations.
  
  
  CONCLUSIONS
  These results indicate that determinants of resistance to ribavirin could also confer resistance to other anti-HCV drugs, shedding light toward understanding the mechanism of action of ribavirin and highlighting the importance of combination drug selection for HCV treatment. The results also show that it is possible to select a 5-Fluorouracil HCV resistant mutant that replicates to levels similar to parental virus when grown in the absence of 5-Fluorouracil.
authors.affiliation: Division of Viral Products, Center for Biologics Evaluation and Research, FDA, Bethesda, Maryland, United States of America.
authors.forename: Kathleen B
authors.initials: KB
authors.lastname: Mihalik
authors.forename: Dino A
authors.initials: DA
authors.lastname: Feigelstock
mesh_terms: D019943:Amino Acid Substitution; D000998:Antiviral Agents / Q000494:pharmacology* / Q000627:therapeutic use; D002460:Cell Line; D003562:Cytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D024882:Drug Resistance, Viral; D005472:Fluorouracil / Q000494:pharmacology; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics*; D019698:Hepatitis C, Chronic / Q000188:drug therapy / Q000821:virology; D006801:Humans; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D009154:Mutation; D012254:Ribavirin / Q000494:pharmacology* / Q000627:therapeutic use; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D014764:Viral Proteins; C513402:2'-C-methylcytidine; D012254:Ribavirin; D003562:Cytidine; D005472:Fluorouracil
references.citation: Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7289-94
references.pmid: 12754380
references.citation: Gastroenterology. 2010 Jul;139(1):154-62.e4
references.pmid: 20303352
references.citation: Curr Opin Pharmacol. 2008 Oct;8(5):522-31
references.pmid: 18835365
references.citation: Antimicrob Agents Chemother. 2006 Oct;50(10):3444-6
references.pmid: 17005827
references.citation: J Virol. 2007 Feb;81(4):2012-24
references.pmid: 17151116
references.citation: Hepatology. 2008 Jan;47(1):306-20
references.pmid: 18161743
references.citation: Gastroenterology. 2007 Mar;132(3):921-30
references.pmid: 17383421
references.citation: N Engl J Med. 2011 Jul 14;365(2):176; author reply 177-8
references.pmid: 21751914
references.citation: J Med Chem. 2006 Nov 2;49(22):6614-20
references.pmid: 17064080
references.citation: Virol J. 2011 Aug 15;8:402
references.pmid: 21843317
references.citation: PLoS One. 2009;4(5):e5554
references.pmid: 19436746
references.citation: J Virol. 2012 Mar;86(5):2869-73
references.pmid: 22190724
references.citation: J Biol Chem. 2005 May 6;280(18):18202-10
references.pmid: 15746101
references.citation: J Virol. 2004 Jul;78(14):7400-9
references.pmid: 15220413
references.citation: Antimicrob Agents Chemother. 2011 Aug;55(8):3854-60
references.pmid: 21628542
references.citation: Nature. 2006 Jan 19;439(7074):344-8
references.pmid: 16327776
references.citation: Nat Struct Biol. 1999 Oct;6(10):937-43
references.pmid: 10504728
references.citation: Biochemistry. 2003 Sep 9;42(35):10462-71
references.pmid: 12950173
references.citation: J Virol. 2013 Jun;87(11):6172-81
references.pmid: 23536652
references.citation: Gastroenterology. 2011 Dec;141(6):2047-55; quiz e14
references.pmid: 21925126
references.citation: J Biol Chem. 2001 Dec 7;276(49):46094-8
references.pmid: 11602568
references.citation: Hepatology. 2012 Mar;55(3):749-58
references.pmid: 22006408
references.citation: Gastroenterology. 2007 May;132(5):1757-66
references.pmid: 17484873
references.citation: Virology. 2003 Jul 5;311(2):339-49
references.pmid: 12842623
references.citation: J Clin Invest. 1995 Jul;96(1):224-30
references.pmid: 7542279
references.citation: J Biol Chem. 2004 Nov 19;279(47):48576-87
references.pmid: 15339921
references.citation: Antivir Ther. 2007;12(5):805-13
references.pmid: 17713164
references.citation: J Virol. 2001 Sep;75(17):8074-81
references.pmid: 11483752
references.citation: Proc Natl Acad Sci U S A. 1990 Sep;87(17):6547-9
references.pmid: 2168552
references.citation: J Gastroenterol. 2007 Oct;42(10):845-53
references.pmid: 17940838
references.citation: Virology. 2006 Aug 1;351(2):349-59
references.pmid: 16713611
references.citation: PLoS One. 2012;7(3):e32550
references.pmid: 22442668
references.citation: J Virol. 2002 Sep;76(17):8505-17
references.pmid: 12163570
references.citation: Science. 2005 Jul 22;309(5734):623-6
references.pmid: 15947137
references.citation: J Virol. 2003 Jan;77(1):481-8
references.pmid: 12477853
references.citation: Nucleosides Nucleotides Nucleic Acids. 2005;24(5-7):767-70
references.pmid: 16248033
references.citation: J Gen Virol. 2004 Sep;85(Pt 9):2485-2502
references.pmid: 15302943
references.citation: Nat Med. 2000 Dec;6(12):1375-9
references.pmid: 11100123
references.citation: N Engl J Med. 2002 Sep 26;347(13):975-82
references.pmid: 12324553
references.citation: J Viral Hepat. 2004 Nov;11(6):479-87
references.pmid: 15500548
references.citation: Antiviral Res. 2005 Jul;67(1):38-45
references.pmid: 15919121
references.citation: Oncologist. 2002;7(4):288-323
references.pmid: 12185293
references.citation: Hepatology. 1993 May;17(5):763-71
references.pmid: 8387945
references.citation: J Virol. 2003 May;77(9):5487-92
references.pmid: 12692249
references.citation: Virol J. 2010 Feb 19;7:44
references.pmid: 20170495
references.citation: N Engl J Med. 2011 Sep 15;365(11):1014-24
references.pmid: 21916639