f The hepatitis C virus (HCV) RNA-dependent RNA-polymerase NS5B is essentially required for viral replication and serves as a prominent drug target. Sofosbuvir is a prodrug of a nucleotide analog that interacts selectively with NS5B and has been approved for HCV treatment in combination with ribavirin. Although the emergence of resistance to sofosbuvir is rarely seen in the clinic, the S282T mutation was shown to decrease susceptibility to this drug. S282T was also shown to confer hypersusceptibility to ribavirin, which is of potential clinical benefit. Here we devised a biochemical approach to elucidate the underlying mechanisms. Recent crystallographic data revealed a hydrogen bond between S282 and the 2=-hydroxyl of the bound nucleotide, while the adjacent G283 forms a hydrogen bond with the 2=-hydroxyl of the residue of the template that base pairs with the nucleotide substrate. We show that DNA-like modifications of the template that disrupt hydrogen bonding with G283 cause enzyme pausing with natural nucleotides. However, the specifically introduced DNA residue of the template reestablishes binding and incorporation of sofosbuvir in the context of S282T. Moreover, the DNA-like modifications of the template prevent the incorporation of ribavirin in the context of the wild-type enzyme, whereas the S282T mutant enables the binding and incorporation of ribavirin under the same conditions. Together, these findings provide strong evidence to show that susceptibility to sofosbuvir and ribavirin depends crucially on a network of interdependent hydrogen bonds that involve the adjacent residues S282 and G283 and their interactions with the incoming nucleotide and complementary template residue, respectively. H epatitis C virus (HCV) has a single-stranded RNA genome of positive polarity and belongs to the Flaviviridae family (1). Chronic HCV infection is associated with severe liver disease, including cirrhosis, and an increased risk of hepatocellular carcinoma (2). If adequately treated, HCV can be cured. Previously, treatment options for HCV infected persons were limited to combination therapy with pegylated alpha interferon (IFN-␣) and ribavirin (3). Ribavirin is a nucleoside analog, with a guanine-like base moiety, that can be incorporated by the HCV RNA-dependent RNA polymerase (RdRp) opposite cytosine or uracil, although the detailed mechanism of action remains elusive (4). In 2011, the first direct-acting antivirals (DAAs) targeting the viral protease, or nonstructural protein 3 (NS3), were approved as a component of IFN-based therapies (5); however, the clinical use of the narrow-spectrum protease inhibitors boceprevir and telaprevir is limited by a narrow coverage of HCV genotypes and a low barrier to the selection of resistance (6). Nucleoside or nucleotide inhibitors (NIs) that target the HCV RdRp, or nonstructural protein 5B (NS5B), address these weaknesses (7). Sofosbuvir, a nucleotide prodrug of 2=-deoxy-2=-␣-fluoro--C-methyluridine, was approved by the U.S. Food and Drug Administration (FDA) in Dece...