A continuous nonradioactive assay for RNA-dependent RNA polymerase activity

被引:19
作者
Lahser, FC [1 ]
Malcolm, BA [1 ]
机构
[1] Schering Plough Res Inst, Dept Antiviral Therapeut, Kenilworth, NJ 07033 USA
关键词
RNA-dependent RNA polymerase; RdRp; coupled-enzyme assay; luciferase; ATP sulfurylase; hepatitis C virus; poliovirus;
D O I
10.1016/j.ab.2003.10.034
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Current assays for the activity of viral RNA-dependent RNA polymerases (RdRps) are inherently end-point measurements, often requiring the use of radiolabeled or chemically modified nucleotides to detect reaction products. In an effort to improve the characterization of polymerases that are essential to the life cycle of RNA viruses and develop antiviral therapies that target these enzymes, a continuous nonradioactive assay was developed to monitor the activity of RdRps by measuring the release of pyrophosphate (PPi) generated during nascent strand synthesis. A coupled-enzyme assay method based on the chemiluminescent detection of PPi. using ATP sulfurylase and firefly luciferase, was adapted to monitor poliovirus 3D polymerase (3D(pol)) and the hepatitis C virus nonstructural protein 513 (NS5B) RdRp reactions. Light production was dependent on RdRp and sensitive to the concentration of oligonucleotide primer directing RNA synthesis. The assay system was found to be amenable to sensitive kinetic studies of RdRps, requiring only 6nM 3D(pol) to obtain a reliable estimate of the initial velocity in as little as 4 min. The assay can immediately accommodate the use of both homopolymer and heteropolymer RNA templates lacking uridylates and can be adapted to RNA templates containing uridine by substituting alpha-thio ATP for ATP. The low background signal produced by other NTPs can be corrected from no enzyme (RdRp) controls. The effect of RdRp/RNA template preincubation was assessed using NS5B and a homopolymer RNA template and a time-dependent increase of RdRp activity was observed. Progress curves for a chain terminator (3'-deoxyguanosine 5'-triphosphate) and an allosteric NS5B inhibitor demonstrated the predicted time- and dose-dependent reductions in signal. This assay should facilitate detailed kinetic studies of RdRps and their potential inhibitors using either standard or single-nucleotide approaches. (C) 2003 Elsevier Inc. All rights reserved.
引用
收藏
页码:247 / 254
页数:8
相关论文
共 37 条
[1]   THE FUNCTION OF COENZYME-A IN LUMINESCENCE [J].
AIRTH, RL ;
RHODES, WC ;
MCELROY, WD .
BIOCHIMICA ET BIOPHYSICA ACTA, 1958, 27 (03) :519-532
[2]   Poliovirus RNA-dependent RNA polymerase (3Dpol) is sufficient for template switching in vitro [J].
Arnold, JJ ;
Cameron, CE .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (05) :2706-2716
[3]   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
[4]   Structure of a transcribing T7 RNA polymerase initiation complex [J].
Cheetham, GMT ;
Steitz, TA .
SCIENCE, 1999, 286 (5448) :2305-2309
[5]   Structural basis for initiation of transcription from an RNA polymerase-promoter complex [J].
Cheetham, GMT ;
Jeruzalmi, D ;
Steitz, TA .
NATURE, 1999, 399 (6731) :80-83
[6]   KINETICS OF FIREFLY LUCIFERASE CATALYZED REACTIONS [J].
DELUCA, M ;
MCELROY, WD .
BIOCHEMISTRY, 1974, 13 (05) :921-925
[7]   Identification and biological characterization of heterocyclic inhibitors of the hepatitis C virus RNA-dependent RNA polymerase [J].
Dhanak, D ;
Duffy, KJ ;
Johnston, VK ;
Lin-Goerke, J ;
Darey, M ;
Shaw, AN ;
Gu, BH ;
Silverman, C ;
Gates, AT ;
Nonnemacher, MR ;
Earnshaw, DL ;
Casper, DJ ;
Kaura, A ;
Baker, A ;
Greenwood, C ;
Gutshall, LL ;
Maley, D ;
DelVecchio, A ;
Macarron, R ;
Hofmann, GA ;
Alnoah, Z ;
Cheng, HY ;
Chan, G ;
Khandekar, S ;
Keenan, RM ;
Sarisky, RT .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2002, 277 (41) :38322-38327
[8]   Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex [J].
Egger, D ;
Wölk, B ;
Gosert, R ;
Bianchi, L ;
Blum, HE ;
Moradpour, D ;
Bienz, K .
JOURNAL OF VIROLOGY, 2002, 76 (12) :5974-5984
[9]   Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli [J].
Ferrari, E ;
Wright-Minogue, J ;
Fang, JWS ;
Baroudy, BM ;
Lau, JYN ;
Hong, Z .
JOURNAL OF VIROLOGY, 1999, 73 (02) :1649-1654
[10]   POLIOVIRUS-SPECIFIC PRIMER-DEPENDENT RNA-POLYMERASE ABLE TO COPY POLY(A) - [RNA-DEPENDENT RNA-POLYMERASE (REPLICASE)-PICORNAVIRUSES] [J].
FLANEGAN, JB ;
BALTIMORE, D .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1977, 74 (09) :3677-3680