Summary.
The hepatitis C virus RNA polymerase (NS5B) is strictly required for viral replication and thus represents an attractive target for antiviral drug development. In this study, stable HeLa cell lines with an integrated NS5B gene were selected by G418 and then confirmed by genome PCR. Subsequently, transcription and expression of the integrated NS5B genes were demonstrated by RT-PCR and Western blot analysis. Further analysis demonstrated enzymatic activity of the expressed NS5B polymerase. The stable HeLa cell lines should be useful for the identification of NS5B inhibitors and for studying the mechanisms of HCV replication.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
T Adam RT Gates BG Sarisky (2004) ArticleTitleSequence requirements for the development of a chimeric HCV replicon system. Virus Res 100 213–222
RH Al Y Wang CH Hagedorn (1998) ArticleTitleExpression of recombinant hepatitis C virus non-structural protein 5B in Escherichia coli. Virus Res 53 141–149 Occurrence Handle10.1016/S0168-1702(97)00147-0 Occurrence Handle1:CAS:528:DyaK1cXhs1Gnsr4%3D Occurrence Handle9620206
SE Behrens L Tomei R De Francesco (1996) ArticleTitleIdentification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J 15 12–22 Occurrence Handle1:CAS:528:DyaK28XlsVKhsA%3D%3D Occurrence Handle8598194
G Chander MS Sulkowski MW Jenckes MS Torbenson HF Bass (2002) ArticleTitleTreatment of chronic hepatitis C: a systematic review. Hepatology 36 S135–S144 Occurrence Handle10.1053/jhep.2002.37146 Occurrence Handle12407587
QL Choo G Kuo AJ Weiner LR Overby DW Bradley M Houghton (1989) ArticleTitleIsolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244 359–362 Occurrence Handle1:CAS:528:DyaL1MXit1Khtrk%3D Occurrence Handle2523562
JK Craggs JK Ball BJ Thomson WL Irving AM Grabowska (2001) ArticleTitleDevelopment of a strand-specific RT-PCR based assay to detect the replicative form of hepatitis C virus RNA. J Virol Methods 94 IssueID1–2 111–120 Occurrence Handle1:CAS:528:DC%2BD3MXjtFGhsrY%3D Occurrence Handle11337045
E Ferrari J Wright-Minogue JW Fang BW Baroudy JY Lau Z Hong (1999) ArticleTitleCharacterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli. J Virol 73 1649–1654 Occurrence Handle1:CAS:528:DyaK1MXmtlamtw%3D%3D Occurrence Handle9882374
L Goobar Larsson L Wiklund S Schwartz (2001) ArticleTitleIntracellular hepatitis C virus RNA-dependent RNA polymerase activity. Arch Virol 146 IssueID8 1553–1570 Occurrence Handle1:CAS:528:DC%2BD3MXnslGntLs%3D Occurrence Handle11676417
A Grakoui C Wychowski C Lin SM Feinstone CM Rice (1993) ArticleTitleExpression and identification of hepatitis C virus polyprotein cleavage products. J Virol 67 1385–1395 Occurrence Handle1:CAS:528:DyaK3sXhs1Crur4%3D Occurrence Handle7679746
AA Kolykhalov EV Agapov KJ Blight K Mihalik SM Feinstone CM Rice (1997) ArticleTitleTransmission of hepatitis C by intrahepatic inoculation with transcribed RNA. Science 277 570–574 Occurrence Handle10.1126/science.277.5325.570 Occurrence Handle1:CAS:528:DyaK2sXkvVCgtbc%3D Occurrence Handle9228008
AA Kolykhalov K Mihalik SM Feinstone CM Rice (2000) ArticleTitleHepatitis c virus-encoded enzymatic activities and conserved RNA elements in the 3′ nontranslated region are essential for virus replication in vivo. J Virol 74 2046–2051 Occurrence Handle10.1128/JVI.74.4.2046-2051.2000 Occurrence Handle1:CAS:528:DC%2BD3cXptlGitA%3D%3D Occurrence Handle10644379
V Lohmann F Korner U Herian R Bartenschlager (1997) ArticleTitleBiochemical properties of hepatitis C Virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity. J Virol 71 8416–8428 Occurrence Handle1:CAS:528:DyaK2sXmvV2hs7s%3D Occurrence Handle9343198
G Luo (1999) ArticleTitleCellular proteins bind to the poly (U) tract of the 3′ untranslated region of hepatitis C virus RNA genome. Virology 256 105–118 Occurrence Handle10.1006/viro.1999.9639 Occurrence Handle1:CAS:528:DyaK1MXhvFWiu7g%3D Occurrence Handle10087231
S Reigadas M Ventura L Sarih-Cottin M Castroviejo S Litvak T Astier Gin (2001) ArticleTitleHCV RNA-dependent RNA polymerase replicates in vitro the 3′ terminal region of the minus-strand viral RNA more efficiently than the 3′ terminal region of the plus RNA. Eur J Biochem 268 5857–5867 Occurrence Handle10.1046/j.0014-2956.2001.02532.x Occurrence Handle1:CAS:528:DC%2BD3MXosFymsLs%3D Occurrence Handle11722573
L Tomei RL Vitale I Incitti S Serafini S Altamura A Vitelli R De Francesco (2000) ArticleTitleBiochemical characterization of a hepatitis C virus RNA-dependent RNA polymerase mutant lacking the c-terminal hydrophobic sequence. J Gen Virol 81 759–769 Occurrence Handle1:CAS:528:DC%2BD3cXhs1Wqtrg%3D Occurrence Handle10675414
InstitutionalAuthorNameWorld Health Organization (1998) ArticleTitleWHO concerns on hepatitis C. Lancet 351 1415
X Zhang H Zhang C Zheng C Li W Xiong (2002) ArticleTitleExtremely low frequency (ELF) pulsed-gradientmagnetic fields inhibit malignant tumour growth at different biological levels. Cell Biol Int 26 599–603 Occurrence Handle1:CAS:528:DC%2BD38XhvVSmu7w%3D Occurrence Handle12127939
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kong, L., Ye, L., Ye, L. et al. Establishment of stable HeLa cell lines expressing enzymatically active hepatitis C virus RNA polymerase. Arch Virol 151, 361–367 (2006). https://doi.org/10.1007/s00705-005-0656-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00705-005-0656-0