Elsevier

Antiviral Research

Volume 89, Issue 2, February 2011, Pages 136-148
Antiviral Research

Hepatitis C virus complete life cycle screen for identification of small molecules with pro- or antiviral activity

https://doi.org/10.1016/j.antiviral.2010.12.005Get rights and content

Abstract

Infection with the hepatitis C virus represents a global public health threat given that an estimated 170 million individuals are chronically infected and thus at risk for cirrhosis and hepatocellular carcinoma. A number of direct antiviral molecules are in clinical development. However, side effects, drug resistance and viral genotype-specific differences in efficacy may limit these novel therapeutics. Therefore, a combination of well tolerated drugs with distinct mechanisms of action targeting different steps of the viral replication cycle will likely improve viral response rates and therapy success.

To identify small molecules that interfere with different steps of the HCV replication cycle, we developed a novel dual reporter gene assay of the complete HCV life cycle and adapted it to 384-well high-throughput format. The system is based on a highly permissive Huh-7 cell line stably expressing a secreted luciferase. Using these cells and an efficient HCV luciferase reporter virus, perturbations of each step of the viral replication cycle as well as cell viability can be easily and quantitatively determined. The system was validated with a selected set of known HCV entry, replication and assembly inhibitors and then utilized to screen a library of small molecules derived from myxobacteria. Using this approach we identified a number of molecules that specifically inhibit HCV cell entry, or primarily virus assembly and release. Moreover, we also identified molecules that increase viral propagation. These compounds may be useful leads for development of novel HCV inhibitors and could be instrumental for the identification of as yet unknown host-derived viral resistance and dependency factors.

Introduction

HCV is a positive-sense, hepatotropic RNA virus of the family Flaviviridae (Lemon et al., 2007). The virus is transmitted parenterally and new infections are primarily acquired by sharing contaminated needles, in healthcare settings and – probably to a lesser extent – through vertical and sexual transmission (Maheshwari et al., 2008, Martinez-Bauer et al., 2008, Deterding et al., 2008). While acute HCV infection is mostly asymptomatic, in 55–85% of cases virus infection is not eliminated (Hoofnagle, 2002) and within 20 years about 20% of chronically infected adults will develop cirrhosis (Seeff, 2002). In fact, chronic HCV infection is one of the most common indications for orthotopic liver transplantation (Brown, 2005). HCV is highly variable and viral strains are classified into at least seven different genetic groups (genotypes, GTs) which differ from each other by ca. 31–33% at the nucleotide level (Simmonds et al., 2005, Gottwein et al., 2009). This enormous variability is a key mechanism that permits continuous viral immune evasion and a substantial challenge for development of antiviral therapy. Chronic HCV infection is treated with pegylated interferon alpha (peg-IFNα) and ribavirin curing 80% of genotype 2 and 3 and approximately 50% of genotype 1-infected individuals (Manns et al., 2006). Given the suboptimal response rates and substantial side effects of this treatment, new direct antiviral inhibitors are being developed and clinically tested (for a recent review, see Schinazi et al., 2010).

The HCV genome is about 9.6 kb in length and encodes a polyprotein of about 3000 amino acids in a single open reading frame. Co- and post-translational cleavages mediated by both cellular and viral proteases liberate at least 10 viral proteins, of which at least the N-terminal three (core, E1 and E2) are major constituents of the extracellular virion (Moradpour et al., 2007), whereas the non-structural proteins NS3 to NS5B are necessary and sufficient for viral RNA replication (Lohmann et al., 1999). The NS2 protease mediates an essential cleavage of the polyprotein at the NS2–NS3 junction, and the p7 protein forms cation-selective ion channels in vitro (Grakoui et al., 1993, Hijikata et al., 1993, Premkumar et al., 2004, Griffin et al., 2003, Pavlović et al., 2003). Both proteins are essential co-factors for the assembly and release of infectious HCV particles (Steinmann et al., 2007a, Steinmann et al., 2007b, Jones et al., 2007).

Upon attachment to the cell surface and utilization of the four minimal cellular entry factors, CD81, scavenger receptor class B type I (SR-BI), claudin-1 (CLDN1) and occludin (OCLN) (Pileri et al., 1998, Scarselli et al., 2002, Evans et al., 2007, Ploss et al., 2009, Liu et al., 2009), the virus is taken up by the cell via clathrin-mediated endocytosis (Blanchard et al., 2006). Subsequently, the RNA genome is released into the cytoplasm in a pH-dependent fusion step that occurs in early endosomes (Tscherne et al., 2006, Koutsoudakis et al., 2006, Meertens et al., 2006) that are acidified by vacuolar ATPases (V-ATPases) (Casey et al., 2010). Important post-fusion steps of the HCV replication cycle are mediated by the cellular microtubule network (Roohvand et al., 2009), which is also involved in the formation and transport of membrane-associated replication complexes designated as membranous webs where RNA replication takes place (Gosert et al., 2003, Wölk et al., 2008). Ultimately, newly synthesized viral RNA is packaged into progeny virus particles, which are then liberated from the infected cell.

Use of HCV cell culture systems including subgenomic replicons (Lohmann et al., 1999), retroviral HCV pseudoparticles (HCVpp) (Bartosch et al., 2003, Hsu et al., 2003) and the JFH1-based infection system (Lindenbach et al., 2005, Wakita et al., 2005, Zhong et al., 2005) has revealed details of the molecular mechanisms that govern HCV replication. Together with recent genome-wide host factor screenings based on RNA-interference, application of these models has identified multiple HCV dependency factors (Li et al., 2009, Tai et al., 2009). Among these, particularly cellular proteins involved in lipid metabolism participate in multiple steps of the viral life cycle (summarized in (Ye, 2007, Popescu and Dubuisson, 2010)): For instance, lipoproteins like HDL and oxidized LDL modulate the efficiency of HCV cell entry (Bartosch et al., 2005, Voisset et al., 2005, Meunier et al., 2005, von Hahn et al., 2006). Moreover, geranylgeranylated lipids provided by the mevalonate pathway are essential for HCV RNA replication (Ye et al., 2003, Kapadia and Chisari, 2005), probably via mediating membrane association of FBL2 and its interaction with HCV NS5A (Wang et al., 2005). Finally, cellular factors involved in the secretion of lipoproteins like the microsomal triglyceride transfer protein (MTP), apolipoprotein B and E (ApoB, ApoE) have been recognized as essential co-factors of HCV particle production (Huang et al., 2007, Chang et al., 2007, Gastaminza et al., 2008). Together these studies highlight a complex and intricate network of HCV dependence on host-derived factors and pathways.

Large-scale screenings for identification of new HCV inhibitors and essential pro- or antiviral host factors have been hampered by the lack of efficient high-throughput assays that encompass the complete viral life cycle. Although a number of models based on subgenomic HCV replicons are available to identify inhibitors of HCV RNA replication (Bourne et al., 2005, Ng et al., 2007, Peng et al., 2007, Mondal et al., 2009), only very recently first assays have been established that monitor interference with other steps of the viral life cycle (Li et al., 2009, Gastaminza et al., 2010, Chockalingam et al., 2010). Here we describe an unbiased cell-based screening system encompassing the entire viral replication cycle that discriminates between antiviral activity and cytotoxicity and at the same time distinguishes between inhibitory influence on RNA translation and replication, and other steps of the viral life cycle. This novel model was used to screen a unique library of natural compounds from myxobacteria thus identifying a number of bioactive compounds with pro- or anti-viral activity and distinct mode of action.

Section snippets

Plasmids

The plasmids pFK-Luc-Jc1 and pFK-Jc1, encoding the genotype 2a/2a chimera Jc1 with or without the firefly luciferase reporter gene, as well as the reporter replicon pFKi389Luc-EI/NS3-3′_JFH1_dg have been described recently (Pietschmann et al., 2006, Koutsoudakis et al., 2006). The plasmids pHIT60 (Cannon et al., 1996), a MLV Gag-Pol expression construct, pRV-F-Luc, a firefly luciferase transducing vector, as well as pczVSV-G (Kalajzic et al., 2001) or pcDNA3ΔcE1E2-J6 or pHIT456 (Cannon et al.,

Establishment and validation of a cell-based assay for screening of the complete HCV replication cycle

To facilitate identification of novel bioactive compounds with HCV-specific antiviral activity, we developed a robust and sensitive cell based assay based on a cell line derived from Huh-7 human hepatocarcinoma cells. The cells used in this study originated from Huh7-Lunet cells that are highly permissive to HCV RNA replication (Friebe et al., 2005), but which express only limited quantities of CD81, an essential HCV entry factor (Koutsoudakis et al., 2006). Robust expression of human CD81 was

Discussion

Only very few cell-based screening systems at high-throughput level are available that permit identification of HCV-specific inhibitors interfering with any step of the viral life cycle. Here we describe the establishment of a 384-well based screening set up that encompasses the complete HCV life cycle. Using a two-step procedure consisting of initial transfection and subsequent inoculation of naïve cells, the assay not only identifies HCV-specific inhibitors but also distinguishes between

Acknowledgements

We are grateful to Takaji Wakita for gift of the JFH1 isolate and to Jens Bukh for the J6CF strain, to Didier Trono for provision of the pWPI, and pCMVΔR8.74 constructs and to Timothey Tellinghuisen for sharing 2′CMA. We would also thank all members of the department of Experimental Virology for helpful suggestions and discussions. This work was supported by an Emmy Noether-fellowship (PI 734/1-1) from the Deutsche Forschungsgemeinschaft (DFG), by grants from the Initiative and Networking Fund

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