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

Abstract

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 (EC 50 s) of 46.8 and 16.1 pM, respectively. Resistance selection studies with the JFH1 replicon and virus systems identified druginduced 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. Secondsite 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.Hepatitis C virus (HCV), a positive-strand RNA virus of the Flaviviridae family, is a leading cause of liver disease and hepatocellular carcinoma (35, 59). The HCV genome encodes a single polyprotein that is cleaved by cellular and viral proteases into at least 10 individual proteins (40, 48). Nonstructural protein 3 (NS3), NS4A, NS4B, NS5A, and NS5B are sufficient for HCV RNA replication in cell culture (6, 30). NS3-4A is the primary viral protease, and NS5B is an RNAdependent RNA polymerase (4,5,14). NS4B, a hydrophobic protein with multiple trans-membrane domains, induces an endoplasmic reticulum-derived membranous web that harbors the HCV replication complex (10, 32). NS5A is also essential for HCV RNA replication, but its precise roles in this process are poorly understood (21, 33).NS5A is an RNA binding protein that interacts with other HCV nonstructural proteins and is capable of altering NS5B polymerase activity in vitro (9,20,41,46,47). NS5A also interacts with a variety of cellular proteins and is potentially involved in modulating multiple aspects of the cellular environment (33, 43). In addition to its role(s) in RNA replication, NS5A is also required for viral assembly (3, 50). NS5A is a modular protein with an N-terminal amphipathic ␣-helix membrane anchor (amino acids [aa] 5 t...

Intragenic Complementation of Hepatitis C Virus NS5A RNA Replication-Defective Alleles

“…Transient replication assays were performed as previously described (26,29). Briefly, replicon transcripts, generated with a Ribomax T7 express system (Promega Corp., Madison, WI) from XbaIlinearized plasmids, were transfected into Huh7.5 cells by using DMRIE-C (liposome formulation of the cationic lipid DMRIE [1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide] and cholesterol) reagent (Invitrogen, Corp., Carlsbad, CA).…”

“…Human hepatoma (Huh-7.5) and baby hamster kidney (BHK1) cells were maintained as previously described (29). A plasmid encoding a bicistronic replicon with the Con1 HCV 5= untranslated region (5=UTR) and internal ribosomal entry site (IRES) sequences, a Renilla luciferase (Rluc) reporter gene, and an encephalomyocarditis virus (EMCV) IRES followed by the NS3 through 3=UTR region from the JFH1 strain has been described (26). NS5A amino acid substitutions were introduced into the replicon plasmid by replacing a BspE1-BspE1 fragment with PCR-generated fragments and were confirmed by sequence analysis.…”

“…As a consequence of the membrane compartmentalization and sequestration of viral replication complexes, diffusion of virus-encoded replication proteins between replication complexes encoded by different viral genomes may be limited, leading to a preference for cis-dominant replication functions. For HCV, cis-acting RNA replication functions have been reported for NS3, NS4A, and NS5B, while transacting functions have been reported for NS5A and NS4B (25)(26)(27)(28).…”

HCV NS5A Inhibitors Disrupt Replication Factory Formation: A Novel Mechanism of Antiviral Action

“…12 In this context, it is noteworthy that preformed replication complexes (RCs) are refractory to inhibition of HCV RNA replication by NS5A inhibitors. [12][13][14] Furthermore, NS5A inhibitors have been shown to induce redistribution of NS5A from endoplasmic reticulum-derived foci, [12][13][14] possibly to lipid droplets, 12 and limit hyperphosphorylation of NS5A, [14][15][16] although these effects may be indirect. Finally, it has been suggested that NS5A inhibitors may disrupt interactions of NS5A with HCV RNA, other viral proteins, and/or host factors that are coopted by NS5A during the HCV life cycle.…”

Model Membrane Platforms for Biomedicine: Case Study on Antiviral Drug Development

“…With the promising results of a recent Phase I clinical trial for the NS5A-targeting BMS-790052 compound against HCV infection in patients who followed a once-daily, dosing regimen [119], there is huge potential for developing additional inhibition strategies against the NS5A protein. Indeed, the amphipathic character of the AH is conserved across all HCV isolates to date [63,64,67], making the AH far less likely to evolve resistance mutations [120,121]. The QCM-D vesicle rupturing assay could be alternatively used to screen chemical libraries for small molecules that inhibit vesicle rupturing by the NS5A AH peptide.…”