Mutations in HCV non-structural genes do not contribute to resistance to nitazoxanide in replicon-containing cells

被引:16
作者
Yon, Changsuek [1 ]
Viswanathan, Prasanth [1 ]
Rossignol, Jean-Francois [2 ,3 ]
Korba, Brent [1 ]
机构
[1] Georgetown Univ, Med Ctr, Dept Microbiol & Immunol, Washington, DC 20007 USA
[2] Romark Inst Med Res, Tampa, FL USA
[3] Stanford Univ, Med Ctr, Dept Med, Div Gastroenterol & Hepatol, Stanford, CA 94305 USA
关键词
Nitazoxanide; Hepatitis C virus; Drug resistance; Replicon; Cell culture; HEPATITIS-C VIRUS; DEPENDENT RNA-POLYMERASE; HELICASE ACTIVITY; SEQUENCE MOTIFS; IN-VITRO; NS3; REPLICATION; REGION; PROTEASE; IDENTIFICATION;
D O I
10.1016/j.antiviral.2011.05.017
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
Nitazoxanide (NTZ) exhibits potent antiviral activity against hepatitis C virus (HCV) in cell culture. Previously, HCV replicon-containing cell lines resistant to NTZ were selected, but transfer the HCV NTZ-resistance phenotype was not observed following transfection of whole cell RNA. To further explore the nature of the resistance of HCV to NTZ, full length HCV replicon sequences were obtained from two NTZ-resistant (NTZ-11, TIZ-9), and the parental (RP7) cell lines. Numerous nucleotide changes were observed in individual HCV genomes relative to the RP7 HCV consensus sequence, but no common mutations in the HCV non-structural genes or 3'-UTR were detected. A cluster of single nucleotide mutations was found within a 5-base portion of the 5'-UTR in 20/21 HCV replicon sequences from both resistant cell lines. Three mutations (5'-UTR G17A, G18A, C20U) were individually inserted into CON1 ('wild-type') HCV replicons, showed reduced replication (5 to 50-fold), but none conferred resistance to NTZ. RP7, NTZ-11, and TIZ-9 were cured of HCV genomes by serial passage under interferon. Transfection of cured NTZ-11 and TIZ-9 with either whole cell RNAs from RP7, NTZ-11, or TIZ-9, 'wild-type' or the 5'-UTR mutation-containing replicon RNAs exhibited an NTZ-resistance phenotype. TIZ (the active metabolite of NTZ) was found to be inactive against the activity of HCV polymerase, protease, and helicase in enzymatic assays. These data confirm previous speculations that HCV resistance to NTZ is not due to mutations in the virus, and demonstrate that HCV resistance and most likely the antiviral activity of TIZ are due to interactions with cellular target(s). (C) 2011 Elsevier B.V. All rights reserved.
引用
收藏
页码:233 / 240
页数:8
相关论文
共 34 条
[1]   Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus [J].
Behrens, SE ;
Tomei, L ;
DeFrancesco, R .
EMBO JOURNAL, 1996, 15 (01) :12-22
[2]   The serine protease domain of hepatitis C viral NS3 activates RNA helicase activity by promoting the binding of RNA substrate [J].
Beran, Rudolf K. F. ;
Serebrov, Victor ;
Pyle, Anna Marie .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2007, 282 (48) :34913-34920
[3]   The hepatitis C virus 3′-untranslated region or a poly(A) tract promote efficient translation subsequent to the initiation phase [J].
Bradrick, SS ;
Walters, RW ;
Gromeier, M .
NUCLEIC ACIDS RESEARCH, 2006, 34 (04) :1293-1303
[4]   SECONDARY STRUCTURE OF THE 5' NONTRANSLATED REGIONS OF HEPATITIS-C VIRUS AND PESTIVIRUS GENOMIC RNAS [J].
BROWN, EA ;
ZHANG, HC ;
PING, LH ;
LEMON, SM .
NUCLEIC ACIDS RESEARCH, 1992, 20 (19) :5041-5045
[5]   SEQUENCE-ANALYSIS OF THE 5' NONCODING REGION OF HEPATITIS-C VIRUS [J].
BUKH, J ;
PURCELL, RH ;
MILLER, RH .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1992, 89 (11) :4942-4946
[6]   Establishment of a simple assay in vitro for hepatitis C virus NS3 serine protease based on recombinant substrate and single-chain protease [J].
Du, GX ;
Hou, LH ;
Guan, RB ;
Tong, YG ;
Wang, HT .
WORLD JOURNAL OF GASTROENTEROLOGY, 2002, 8 (06) :1088-1093
[7]   Amphipathic helix-dependent localization of NS5A mediates hepatitis C virus RNA replication [J].
Elazar, M ;
Cheong, KH ;
Liu, P ;
Greenberg, HB ;
Rice, CM ;
Glenn, JS .
JOURNAL OF VIROLOGY, 2003, 77 (10) :6055-6061
[8]   The Anti-Hepatitis C Agent Nitazoxanide Induces Phosphorylation of Eukaryotic Initiation Factor 2α Via Protein Kinase Activated by Double-Stranded RNA Activation [J].
Elazar, Menashe ;
Liu, Michael ;
Mckenna, Sean A. ;
Liu, Ping ;
Gehrig, Elizabeth A. ;
Puglisi, Joseph D. ;
Rossignol, Jean-Francois ;
Glenn, Jeffrey S. .
GASTROENTEROLOGY, 2009, 137 (05) :1827-1835
[9]  
Frick DN, 2010, FASEB J, V24
[10]   Mutations in hepatitis C virus genotype 1b and the sensitivity of interferon-ribavirin therapy after liver transplantation [J].
Fukuhara, Takasuke ;
Taketomi, Akinobu ;
Okano, Shinji ;
Ikegami, Toru ;
Soejima, Yuji ;
Shirabe, Ken ;
Maehara, Yoshihiko .
JOURNAL OF HEPATOLOGY, 2010, 52 (05) :672-680