Fast Hepatitis C Virus RNA Elimination and NS5A Redistribution by NS5A Inhibitors Studied by a Multiplex Assay Approach

Abstract

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...

NS5A Promotes Constitutive Degradation of IP3R3 to Counteract Apoptosis Induced by Hepatitis C Virus

“…To this end, we used BMS-790052 (daclatasvir), a powerful anti-HCV drug that has been shown to change NS5A localization from ER membranes to lipid droplets (Liu et al, 2015). Thus, we examined the effect of BMS-790052 on NS5A binding to FBXL2 in Huh7.5-JFH1 cells stably expressing FBXL2 under the control of a weak retroviral promoter.…”

Approved Antiviral Drugs over the Past 50 Years

“…12, daclatasvir can specifically bind to the HCV nonstruc-tural protein NS5A (282). The exact mechanisms of HCV NS5A drug action remain debated, especially regarding their potential inhibition of the structural stability, dimerization, or subcellular distribution of NS5A (283,284). Nevertheless, NS5A inhibitors can block HCV RNA replication by interrupting the formation of the membranous web, a heterogeneous meshwork within cytoplasmic membranous factories where HCV replication takes place (284).…”

Modeling how reversal of immune exhaustion elicits cure of chronic hepatitis C after the end of treatment with direct‐acting antiviral agents

“…The residual, predominantly non‐infectious virions would be cleared eventually, leading to SVR. Evidence for this hypothesis comes from studies that show a reduction in viral infectivity following exposure to DAAs, including a preferential targeting of infectious over non‐infectious virion production by some DAAs 15, 16, 17. Furthermore, mathematical models of viral kinetics that account for this mode of DAA action have been constructed and shown to describe data of viral load changes during therapy in patients who experience EOT + /SVR, as well as SVR rates elicited by certain DAA combinations 18, 19.…”