As a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied the in vitro pharmacology of a novel C-nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concentration [EC 50 ], 0.048 to 0.68 M). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC 50 , 1.5 M). The active 5=-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, with K i /K m values of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, respectively. With its unique dual substitutions of 1=-CN and 2=-C-Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistance in vitro. Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymatic assays (>30-fold). Consistent with its in vitro profile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clinical trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability. The recent regulatory approval of two HCV nonstructural protein 3/4A (NS3/4A) protease inhibitors, telaprevir and boceprevir, has led to increased treatment response rates when given in combination with pegylated interferon (IFN) and ribavirin (RBV) for those with an HCV genotype 1 infection. However, these regimens are limited by the emergence of viral resistance, increased adverse events, inability to treat patients who are intolerant or contraindicated to IFN treatment, and decreased efficacy in many patient populations who are most in need of therapy, including those with advanced liver diseases and those infected with other HCV genotypes (4). Nucleotide inhibitors (NIs) have demonstrated great promise as direct-acting antivirals with broad genotype coverage, lack of preexisting variants with reduced susceptibility, a high barrier to resistance, and the ability to produce potent and durable antiviral responses (5-7). Following intrahepatic activation involving nucleotide kinases, the active 5=-triphosphates of NIs target the HCV nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase by serving as alternative substrates and nonobligate chain terminators of viral RNA synthesis. The initial HCV NIs to enter clinical trials were N-nucleosides, containing the natural C-N glycosidic linkage, with either 2=-C-Me or 4=-azido...