Inhibition of hepatitis C virus RNA replication by 2'-modified nucleoside analogs

• Published in: The Journal of biological chemistry 2003-04, Vol.278 (14), p.11979-11984
• Main Authors: Carroll, Steven S, Tomassini, Joanne E, Bosserman, Michele, Getty, Krista, Stahlhut, Mark W, Eldrup, Anne B, Bhat, Balkrishen, Hall, Dawn, Simcoe, Amy L, LaFemina, Robert, Rutkowski, Carrie A, Wolanski, Bohdan, Yang, Zhucheng, Migliaccio, Giovanni, De Francesco, Raffaele, Kuo, Lawrence C, MacCoss, Malcolm, Olsen, David B
• Format: Article
• Language: English
• Subjects: Adenosine - analogs & derivatives; Adenosine - chemistry; Adenosine - pharmacology; Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - chemistry; Cells, Cultured; Cytidine - analogs & derivatives; Cytidine - pharmacology; Cytidine Triphosphate - analogs & derivatives; Cytidine Triphosphate - chemistry; DNA Polymerase beta - antagonists & inhibitors; DNA Polymerase gamma; DNA Polymerase I - antagonists & inhibitors; DNA-Directed DNA Polymerase; Gels; Hepacivirus - genetics; Hepacivirus - growth & development; Hepatitis C - virology; Humans; Nucleic Acid Synthesis Inhibitors; RNA, Viral - genetics; Viral Nonstructural Proteins - antagonists & inhibitors; Viral Nonstructural Proteins - metabolism; Virus Replication - drug effects
• ISSN: 0021-9258
• DOI: 10.1074/jbc.m210914200

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2'-substituted nucleosides as inhibitors of HCV replication. The 5'-triphosphates of 2'-C-methyladenosine and 2'-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2'-O-methylcytidine inhibit HCV RNA replication. The 5'-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2'-C-methyladenosine triphosphate than 2'-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2'-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitro enzyme assays. Thus, the 2'-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication.