e While earlier therapeutic strategies for the treatment of hepatitis C virus (HCV) infection relied exclusively on interferon (IFN) and ribavirin (RBV), four direct-acting antiviral agents (DAAs) have now been approved, aiming for an interferon-free strategy with a short treatment duration and fewer side effects. To facilitate studies on the mechanism of action (MOA) and efficacy of DAAs, we established a multiplex assay approach, which employs flow cytometry, a Gaussia luciferase reporter system, Western blot analysis, reverse transcription-quantitative PCR (RT-qPCR), a limited dilution assay (50% tissue culture infectious dose [TCID 50 ]), and an image profiling assay that follows the NS5A redistribution in response to drug treatment. We used this approach to compare the relative potency of various DAAs and the kinetics of their antiviral effects as a potential preclinical measure of their potential clinical utility. We evaluated the NS5A inhibitors ledipasvir (LDV) and daclatasvir (DCV), the NS3/4A inhibitor danoprevir (DNV), and the NS5B inhibitor sofosbuvir (SOF). In terms of kinetics, our data demonstrate that the NS5A inhibitor LDV, followed closely by DCV, has the fastest effect on suppression of viral proteins and RNA and on redistribution of NS5A. In terms of MOA, LDV has a more pronounced effect than DCV on the viral replication, assembly, and infectivity of released virus. Our approach can be used to facilitate the study of the biological processes involved in HCV replication and help identify optimal drug combinations. Hepatitis C virus (HCV) infects approximately 3% of the world's population, which accounts for about 170 million chronically infected individuals. Annually, there are more than 350,000 deaths from HCV-related cirrhosis and hepatocellular carcinoma (1). In the United States, there are more than 3 million people with chronic HCV infection, and about 15,000 die from HCV-related liver disease each year.HCV is a positive-strand RNA virus grouped in the genus Hepacivirus within the family Flaviviridae (2). It is classified into at least 6 genotypes (gt), and its error-prone polymerase leads to more than 50 subtypes (3). The long open reading frame, which encodes the HCV polyprotein, is processed by host and viral proteases and gives rise to three structural proteins (the capsid protein core and envelope glycoproteins E1 and E2) and seven nonstructural (NS) proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (4). NS2 and p7 are essential for virus assembly but not RNA replication, whereas NS3 to NS5B are involved in a membrane-associated RNA replicase complex (RC) (5). The NS3 protein is composed of a serine protease and an RNA helicase/nucleoside triphosphatase (NTPase), NS4A serves as a cofactor for NS3 serine protease (6), NS5B is the RNA-dependent RNA polymerase (7), and NS5A is considered to play key roles in multiple steps of the HCV life cycle.NS5A is an ϳ450 amino acid phosphoprotein composed of an N-terminal amphipathic ␣-helix and three domains (domain I to domain III), each of wh...

Several direct-acting antivirals (DAAs) are available, providing interferon-free strategies for a hepatitis C cure. In contrast to DAAs, host-targeting agents (HTAs) interfere with host cellular factors that are essential in the viral replication cycle; as host genes, they are less likely to rapidly mutate under drug pressure, thus potentially exhibiting a high barrier to resistance, in addition to distinct mechanisms of action. We compared the effects of cyclosporin A (CsA), a HTA that targets cyclophilin A (CypA), to DAAs, including inhibitors of nonstructural protein 5A (NS5A), NS3/4A, and NS5B, in Huh7.5.1 cells. Our data show that CsA suppressed HCV infection as rapidly as the fastest-acting DAAs. CsA and inhibitors of NS5A and NS3/4A, but not of NS5B, suppressed the production and release of infectious HCV particles. Intriguingly, while CsA rapidly suppressed infectious extracellular virus levels, it had no significant effect on the intracellular infectious virus, suggesting that, unlike the DAAs tested here, it may block a post-assembly step in the viral replication cycle. Hence, our findings shed light on the biological processes involved in HCV replication and the role of CypA.