Distinct Functions of NS5A in Hepatitis C Virus RNA Replication Uncovered by Studies with the NS5A Inhibitor BMS-790052

BMS-790052, targeting nonstructural protein 5A (NS5A), is the most potent hepatitis C virus (HCV) inhibitor described to date. It is highly effective against genotype 1 replicons and also displays robust genotype 1 anti-HCV activity in the clinic (M. Gao et al., Nature 465:96-100, 2010). BMS-790052 inhibits genotype 2a JFH1 replicon cells and cell culture infectious virus with 50% effective concentrations (EC50s) of 46.8 and 16.1 pM, respectively. Resistance selection studies with the JFH1 replicon and virus systems identified drug-induced mutations within the N-terminal region of NS5A. F28S, L31M, C92R, and Y93H were the major resistance mutations identified; the impact of these mutations on inhibitor sensitivity between the replicon and virus was very similar. The C92R and Y93H mutations negatively impacted fitness of the JFH1 virus. Second-site replacements at NS5A residue 30 (K30E/Q) restored efficient replication of the C92R viral variant, thus demonstrating a genetic interaction between NS5A residues 30 and 92. By using a trans-complementation assay with JFH1 replicons encoding inhibitor-sensitive and inhibitor-resistant NS5A proteins, we provide genetic evidence that NS5A performs the following two distinct functions in HCV RNA replication: a cis-acting function that likely occurs as part of the HCV replication complex and a trans-acting function that may occur outside the replication complex. The cis-acting function is likely performed by basally phosphorylated NS5A, while the trans-acting function likely requires hyperphosphorylation. Our data indicate that BMS-790052 blocks the cis-acting function of NS5A. Since BMS-790052 also impairs JFH1 NS5A hyperphosphorylation, it likely also blocks the trans-acting function.