Abstract
The urgent need for efficacious drugs to treat chronic hepatitis C virus (HCV) infection requires a concerted effort to develop inhibitors specific for virally encoded enzymes. We demonstrate that 2'-C-methyl ribonucleosides are efficient chain-terminating inhibitors of HCV genome replication. Characterization of drug-resistant HCV replicons defined a single S282T mutation within the active site of the viral polymerase that conferred loss of sensitivity to structurally related compounds in both replicon and isolated polymerase assays. Biochemical analyses demonstrated that resistance at the level of the enzyme results from a combination of reduced affinity of the mutant polymerase for the drug and an increased ability to extend the incorporated nucleoside analog. Importantly, the combination of these agents with interferon-alpha results in synergistic inhibition of HCV genome replication in cell culture. Furthermore, 2'-C-methyl-substituted ribonucleosides also inhibited replication of genetically related viruses such as bovine diarrhea virus, yellow fever, and West African Nile viruses. These observations, together with the finding that 2'-C-methyl-guanosine in particular has a favorable pharmacological profile, suggest that this class of compounds may have broad utility in the treatment of HCV and other flavivirus infections.
Highlights
Hepatitis C virus (HCV)1 is the most common blood-borne infection and a major cause of chronic liver disease and liver transplantation in industrialized countries
The studies presented demonstrate that 2Ј-C-Memodified nucleosides act as direct inhibitors of HCV NS5B both in vitro and in vivo, validating this compound class as bona fide HCV antiviral agents
To gain further insight into the mechanism of chain termination and resistance, a model of the NS5B initiation complex was generated from the NS5B crystal structure and the ⌽6 RNA-dependent RNA polymerase (RdRp) initiation complex crystal structure (6, 23)
Summary
HBI10A cells were plated in 15-cm tissue culture dishes at a density of 3 ϫ 103/cm and cultured in the presence of 0.8 mg/ml G418 and 2Ј-C-Me-A at concentrations increasing from 1 to 5 M. The NS5B protein was preincubated with template RNA for 15 min at room temperature, and elongation reaction was started by the addition of 100 M UTP, CTP, and GTP and increasing amounts of ATP and [␣-32P]ATP (0.06 Ci/M cold ATP). Elongation was allowed to proceed for 5 min at 37 °C, and the activity was measured as the radioactivity present in the acid-insoluble material Under these conditions, reaction rates at the highest concentration of ATP approached saturation. Reactions were initiated by the addition of a mixture of NTPs and inhibitor after preincubation of the enzyme and template for 30 min at room. Replicase Assay—HCV replication complexes were isolated by differential centrifugation from HB1 or 2Ј-C-Me-A-resistant HB1 cells and assayed as described previously (14) in the presence or absence of 2Ј-C-Me triphosphates.
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