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1 RAPID COMMUNICATION Cyclosporine A Inhibits Hepatitis C Virus Nonstructural Protein 2 Through Cyclophilin A Sandra Ciesek, 1,2 Eike Steinmann, 2 Heiner Wedemeyer, 1 Michael P. Manns, 1 Johann Neyts, 3 Norbert Tautz, 4 Vanesa Madan, 5 Ralf Bartenschlager, 5 Thomas von Hahn, 1,2 and Thomas Pietschmann 2 Numerous anti-hepatitis C virus (HCV) drugs targeting either the viral nonstructural 3 (NS3) protease or NS5B polymerase are currently in clinical testing. However, rapid resistance development is a major problem and optimal therapy will clearly require a combination of multiple mechanisms of action. Cyclosporine A (CsA) and its nonimmunosuppressant derivatives are among the more promising drugs under development. Based on work with subgenomic HCV replicons it has been thought that they act as NS5B-inhibitors. In this study we show that CsA inhibits replication of full-length HCV Japanese Fulminant Hepatitis (JFH1) genomes about 10-fold more efficiently than subgenomic replicons. This effect is dependent on the presence of NS2 in the viral polyprotein and mediated through cellular cyclophilin A. NS2 is either an additional target for CsA-dependent inhibition or modulates the antiviral activity against NS3 to NS5B proteins. CsA is thus the first anti-hcv drug shown to act through NS2. Conclusion: CsA inhibits replication of JFH1 full-length genomes much more efficiently than subgenomic replicons by targeting cleavage at the NS2/NS3 junction and possibly other nonreplication lifecycle steps. (HEPATOLOGY 2009;50: ) Worldwide more than 130 million people are chronically infected with hepatitis C virus (HCV), a highly variable enveloped RNA virus of the Flaviviridae family, that causes chronic liver Abbreviations: CsA, cyclosporine A; CyP, cyclophilin; HCV, hepatitis C virus; IRES, internal ribosome entry site; NS, nonstructural. From the 1 Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Germany; 2 Division of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI); 3 Rega Institute for Medical Research, KU Leuven, Leuven, Belgium; 4 Department of Virology and Cell Biology, University of Lübeck, Germany; 5 Department of Molecular Virology, University of Heidelberg, Germany. Received June 15, 2009; accepted August 24, S.C. was supported by IFB-TX, a program of MHH funded by the Deutsche Forschungs-gemeinschaft, an intramural young investigator award of the HILF initiative, and by a grant from the women support program of the Hannover Medical school. Novartis and DebioPharm supported the study by providing reagents. T.P. was supported by grants from the Helmholtz Association SO-024 and HA-202. Address reprint requests to: Prof. Dr. rer. nat. Thomas Pietschmann, Division of Experimental Virology, TWINCORE Center for Experimental and Clinical Infection Research, Feodor-Lynen-Stra e 7-9, Hannover, Germany. thomas.pietschmann@twincore.de; fax: Copyright 2009 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI /hep Potential conflicts of interest: Dr. Ciesek received grants from Novartis. Dr. Wedemeyer advises, is on the speakers bureau, and grants Novartis. Dr. Manns is a consultant, advises, is on the speakers bureau of, and received grants from Novartis. He also received grants from Debio. Additional Supporting Information may be found in the online version of this article. diseases, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. 1 The HCV genome encodes a single polyprotein that is cleaved into at least 10 viral proteins: the structural proteins (core, E1, E2) that constitute the virion, the p7 ion channel, and six nonstructural (NS) proteins (NS2, 3, 4A, 4B, 5A, and 5B). NS3-5B are sufficient to sustain RNA replication. 2 NS2 is a cysteine protease that cleaves the NS2-3 junction and requires an N-terminal part of NS3 as a cofactor and plays a crucial but vaguely defined role during virus assembly. 3,4 Recently, a cell culture system, based on the Japanese Fulminant Hepatitis (JFH1) HCV isolate, which reproduces the complete viral replication cycle in vitro, was developed. 5 In this study we used this system to assess the impact of CsA on HCV RNA replication, virus production, and infectivity. The immunosuppressive drug cyclosporine A (CsA) has been shown to be active against HCV in vitro and in vivo. 6,7 The two CsA-derivatives Debio-025 and NIM811 lacking anticalcineurin activity (determinant for the immunosuppressive effect of CsA) are now in human trials as HCV inhibitors, with initial data showing good activity and no signs of clinically relevant resistance. 8,9 The antiviral effect of CsA and cyclosporine derivatives were linked to their ability to interact with cyclophilins (CyPs), which in turn were recognized as essential cellular cofactors for HCV. 10,11 CyPs are part of 1638

2 HEPATOLOGY, Vol. 50, No. 5, 2009 CIESEK ET AL a family of cellular peptidyl-prolyl-isomerases. Because in subgenomic HCV replicons, minimal replicating genomes comprising NS3-NS5B, various viral CsA-resistance mutations were found to map to NS5B and cyclophilin B (CypB) was found to associate with NS5B and to stimulate its RNA binding activity, it is believed that NS5B is the primary target of CsA. 12,13 However, standard subgenomic replicons do not express HCV proteins core, E1, E2, p7, and NS2. Thus, it is unclear if the function of any of these proteins is also affected by CsA or if these proteins modulate the antiviral effect of CsA. Our results reveal that CsA inhibits replication of JFH1-derived full-length genomes much more efficiently than subgenomic replicons. We show that NS2 is an additional and possibly more important target for CsA- and Debio-025 than NS5B and that CsA targets NS2 by way of cyclophilin A (CypA). Materials and Methods Drugs. Cyclosporine A was provided by Novartis (Basel, Switzerland). Tacrolimus and interferon (IFN) alpha were purchased from Sigma-Aldrich (Seelze, Germany). Debio-025 (D-MeAla 3 -EtVal 4 -cyclosporine) was provided by Debiopharm (Lausanne, Switzerland). The NS5B polymerase inhibitors JT-16 and 4-azidocytidine were synthesized by Prof. Herdewijn (Leuven, Belgium). 2-C-methyladenosine (2-CMA) was a gift from Tim Tellinghuisen (Jupiter, FL). Plasmids. Construction of the reporter virus genome Luc-Jc1, Luc-JFH1 NS3-5B, Luc-JFH1, Luc-JFH1/ p7, and Luc-JFH1/ E1,E2 have been described recently. 14 Individual genomes used are shown in Figs. 1A, 3A, and 4A. Detailed cloning strategies are available on request. HCV Replication Assay. HCV replication assay was performed as described. 14 Four hours after transfection, different inhibitors were added to the cell culture medium. Quantitative Detection Core Protein. HCV core protein was measured using an HCV core antigen kit (Wako Chemicals, Neuss, Germany). Stable Knockdown and CypA Rescue Cell Lines. For CypA and CypB knockdown, microrna-based short hairpin RNA (shrna) lentiviral vectors were used encoding the puromycin resistance gene and an shrna targeting CypA, CypB, or a control shrna targeting luciferase. The shrna targeting sequences were: luciferase, 5 -tacaaacgctctcatcgacaag-3 (not present in the luciferase gene of the reporter replicon and reporter virus), CypA, 5 -ctggattgcagagttaagttta-3 ; and CypB, 5 -gccgggtgatctttggtctctt-3. For rescue of CypA expression, CypA knockdown cells were transduced with lentiviral vectors expressing wildtype or isomerase-defective CypA (H126Q mutant). Detailed sequence information of these vectors is available on request. Antibodies. For immunoblotting, commercial antibodies against CypA (antimouse, monoclonal; Abcam, Cambridge, UK) and CypB (antirabbit, polyclonal; Dianova, Hamburg, Germany) were used. Results Cyclosporine A and Debio-025 Inhibit Replication of a JFH1-Derived Full-Length Genome Much More Efficiently than a Subgenomic Replicon. To evaluate the effects of CsA on HCV replication and infectivity, we used firefly luciferase reporter viruses (Fig. 1A). Huh-7.5 cells were transfected with the reporter virus genome and 4 hours later CsA was added with increasing doses. RNA replication efficiency was assessed 48 hours after transfection using luciferase assays. In parallel, the supernatant was used to inoculate naïve cells in order to detect a possible influence of the drugs on virus production and infectivity. High concentrations of CsA decreased RNA replication of both the subgenomic replicon and full-length virus by about 4 logs. Sensitivity of the replicon toward CsA was similar to what has been reported. 15 However, in our side-by-side comparison full-length virus showed a 10- fold lower median inhibitory concentration (IC 50 ) (0.15 g/ml versus 1 g/ml), indicating a markedly increased sensitivity to CsA-inhibition (Fig. 1B). When infection of supernatant-inoculated cells was analyzed the observed IC 50 was another 30-fold lower at g/ml, suggesting that CsA may inhibit other viral lifecycle steps in addition to replication (Fig. 1C). Debio-025, a nonimmunosuppressive cyclosporine derivative that does not display calcineurin affinity, showed a similar difference when comparing subgenomic RNA and Jc1 full-length RNA (Fig. 1D). In Contrast to CsA, IFN and Different NS5B Polymerase Inhibitors Block Subgenomic and Full-Length Genomes with Comparable Efficiency and IC 50 Does Not Differ Between Replication and Infectivity. To ensure that the differential susceptibility to CsA is not solely attributable to dissimilar RNA replication efficiency of the full-length genome and subgenomic replicons, we next compared inhibition of HCV RNA replication by other HCV inhibitors including IFN alpha and three NS5B polymerase inhibitors, JT-16, 4-azidocytidine, and 2-CMA. As shown in Fig. 2, we observed similar IC 50 values for the infectious model and the subgenomic replicon in all cases (Fig. 2A-D). Moreover, RNA replication and infectivity in supernatant were equally sensitive to 2-CMA inhibition (Fig. 2D,E).

3 1640 CIESEK ET AL. HEPATOLOGY, November 2009 Fig. 1. Differential effect of cyclosporine A and Debio-025 on HCV RNA replication and virus infection. (A) Schematic representation of the constructs used. J6CF-derived genome segments are depicted in dark gray, JFH1-derived portions are light gray. (B) HCV RNA replication in Huh-7.5 cells 48 hours after transfection with Jc1-Luc or subgenomic RNA. CsA was added 4 hours after transfection. (C) Culture fluid of these cells was used to inoculate naïve Huh-7.5 cells. (D) Inhibition of RNA replication by Debio-025. These data indicate that direct NS5B inhibitors, different from CsA, have no additional inhibitory effects on fulllength genomes and on lifecycle steps other than RNA replication. Increased Sensitivity to CsA Is Associated with HCV NS2. To identify the putative additional CsA target in full-length virus, different HCV genomes with deletions of the structural proteins core, E1, and E2 or p7 as well as a deletion of the entire structural region were constructed (Fig. 3A). All of these behaved like full-length virus, i.e., were inhibited with a 5-fold to 10-fold lower IC 50 compared to a replicon encoding NS3-5B only (Fig. 3B,C). Thus, addition of NS2 alone to a subgenomic replicon is sufficient to confer enhanced CsA sensitivity, indicating that NS2 is the additional target for CsA-dependent inhibition. The Additional Inhibitory Effect of CsA Is Detectable Only When Replication Depends on Cleavage of the NS2/NS3 Junction. To investigate the interplay between CsA and NS2-3 protease function, full-length genomes with an internal ribosome entry site (IRES) between p7/ns2, NS2/NS3, respectively, were created (Fig. 4A). In case of the former, HCV RNA replication depends on a functional NS2-3 protease cleaving at the NS2/3 site, whereas for the latter, RNA replication is uncoupled from this enzymatic activity. As shown in Fig. 4B, insertion of an IRES between NS2 and NS3 lowered CsA sensitivity more than 10-fold with regard to RNA

4 HEPATOLOGY, Vol. 50, No. 5, 2009 CIESEK ET AL Fig. 2. IFN alpha and different polymerase inhibitors decrease replication of subgenomic and fulllength genomes with comparable efficiency. (A-D) Inhibition of RNA replication by interferon and three different specific NS5B polymerase inhibitors. (E) Inoculation of Huh-7.5 cells with supernatant from the two C-methyladenosine-treated cells shown in Fig. 2D. replication, release of core, and infectivity. Thus, the presence of NS2 causes enhanced CsA-sensitivity only when replication depends on cleavage of the NS2/3 junction, suggesting that the effect may be associated with NS2-3 protease function. To rule out that disruption of the RNA nucleotide sequence at the NS2/NS3 junction alone decreases susceptibility to CsA, we confirmed that a genome encoding NS2 and the NS3 protease domain and thus maintaining the NS2-3 junction in front of the IRES also displays lower susceptibility compared to genomes that require NS2/NS3 cleavage for replication (Fig. 4C). The Additional Inhibitory Effect of CsA Is Independent from Calcineurin but Depends on Cyclophilin A. The complex of CyP and the immunosuppressive drug cyclosporine A binds to and inhibits calcineurin, a cellular phosphatase and a key mediator of T-cell activation. To investigate if the increased sensitivity to CsA is mediated by calcineurin, we measured HCV RNA replication in the presence of the cyclophilin independent calcineurin inhibitor tacrolimus. We observed no effect on HCV RNA replication of either subgenomic or full-length genomes (Fig. 5A). To further characterize whether CsA acts on NS2 by way of CyPs we used Huh7-Lunet cells with a stable knockdown of CypA or CypB. As shown in Fig. 5B, 72 hours posttransfection of given HCV constructs into the individual cell lines, we only observed inhibition of HCV RNA replication in CypA knockdown cells for genomes where NS2 is part of the polyprotein (Jc1 and JFH NS2-5B). In contrast, replication of the sub-

5 1642 CIESEK ET AL. HEPATOLOGY, November 2009 Fig. 3. Increased sensitivity to CsA is independent of HCV proteins core, E1, E2, and p7, but associated with HCV NS2. (A) Schematic representation of used constructs. (B,C) Inhibition of RNA replication of the above recombinant HCV genomes by CsA. genomic replicon (NS3-5B) and the full-length construct where an interposed IRES separates NS2 from NS3 was not impaired. These results support that CsA targets NS2 by way of CypA, possibly through interference with NS2-3 cleavage. Peptidyl-prolyl-Isomerase Activity of Cyclophilin A Is Important for NS2/3 Protease Function. One major function of CyPs is to catalyze cis-trans peptidyl-prolyl isomerization during protein folding by way of their peptidyl-prolyl-isomerase activity. Therefore, CyP inhibition may prevent adequate peptidyl-prolyl isomerization of the NS2-NS3 protease, precluding correct protein folding and in turn NS2-3 protease activity. To investigate if the peptidyl-prolyl-isomerase activity of CypA is important for NS2-NS3 protease function, we restored CypA expression in Huh7-Lunet CypA knockdown cells either with wildtype CypA or with a CypA mutant (H126Q) that possesses less than 1% of wildtype isomerase activity. 16 As shown in Fig. 6, efficient replication of the Luc- Jc1 full-length genome and the NS2-5B replicon was only restored when wildtype CypA was introduced, but not with the H126Q mutant. These results argue that the isomerase activity of CypA is essential for efficient replication of HCV genomes that depend on NS2/NS3 cleavage for RNA replication. Discussion In this study we show that CsA and derivatives inhibit HCV infection in a multifold manner: Besides the described effects on NS5B function that have been characterized by others using mostly subgenomic replicons, 8,11-13,17,18 we found a markedly enhanced sensitivity to CsA in full-length virus. Replication of fulllength genomes is inhibited more strongly and there is an additional inhibitory CsA effect on other viral life-

6 HEPATOLOGY, Vol. 50, No. 5, 2009 CIESEK ET AL Fig. 4. The additional inhibitory effect of CsA depends on NS2/3 protease. (A) A set of constructs with an IRES at different positions of the genome was constructed to assess the role of NS2/NS3 cleavage dependent RNA replication on CsA susceptibility. (B) Huh-7.5 cells were transfected and treated with the given quantity of CsA. At 48 hours RNA replication (left), core release (middle), and release of infectivity (right) was assessed. (C) Huh- 7.5 cells were transfected and treated with different amounts of CsA; 48 hours later, HCV RNA replication was measured. cycle steps, possibly assembly and/or release. Furthermore, we determined that enhanced sensitivity is dependent on the presence of viral NS2 and cellular CypA possessing active isomerase activity. These findings have two important implications. First, the success of future therapeutic regimens will likely depend on the ability to combine multiple antiviral mechanisms. The current nonspecific standard substances, pegylated interferon and ribavirin, are expected to be complemented with specific antivirals under development, with inhibitors of NS3 and NS5B being the currently most advanced substances. NS2 has long been seen as an attractive target, but promising lead compounds have not been identified so far. In the light of the data shown here it is conceivable that CsAderivatives may close this gap. In fact, considering the much lower IC 50 of CsA against NS2-containing and full-length viral genomes, NS2 inhibition at least in the context of JFH1-based genomes may be the dominant antiviral effect of CsA-derivatives. In line with this interpretation, knockdown of endogenous CypA only markedly inhibited replication of JFH1 genomes that depend on proper NS2/NS3 cleavage for replication, whereas constructs that replicate independent of this cleavage were not impaired. These results likely reflect the different susceptibility of these constructs to CsA and suggest that CypA abundance in the knockdown cells was rate-limiting only for the NS2-dependent genomes; however, still sufficient for efficient replication of those genomes independent of processing at the NS2/NS3 site. Second, our findings have implications for our understanding of HCV biology: The NS2-dependent enhanced CsA sensitivity of full-length virus is independent of calcineurin, but mimicked by knockdown of CypA, the major cellular cyclophilin family member. Thus, CypA is a critical host factor that serves multiple functions for HCV. NS2 led to enhanced CsA

7 1644 CIESEK ET AL. HEPATOLOGY, November 2009 Fig. 5. The additional inhibitory effect of CsA on NS2 depends on cyclophilin A. (A) Inhibition of HCV RNA replication by tacrolimus. (B) Huh7-Lunet cells stably expressing shrna against CypA or CypB and control cells were transfected with given constructs. HCV RNA replication was measured 72 hours posttransfection. (C) CypA or CypB knockdown efficiency in Huh7-Lunet cells expressing shrna against CypA or CypB was confirmed by western blotting. Fig. 6. The isomerase activity of CypA is important for NS2 function. (A) CypA expression in Lunet CypA knockdown cells was restored using wildtype CypA or a mutant lacking isomerase activity (H126Q) and replication of given constructs was assessed upon transfection. (B) Restoration of CypA expression was confirmed by western blotting.

8 HEPATOLOGY, Vol. 50, No. 5, 2009 CIESEK ET AL sensitivity only if it was translated as part of the viral polyprotein, whereas NS2 translated from a separate cistron had no effect. Moreover, disruption of the RNA sequence of the NS2/NS3 junction alone was not sufficient to confer decreased CsA sensitivity. This is suggestive that the NS2 protease, which mediates a single cleavage event between NS2 and NS3 that is required for liberation of the NS3 N-terminus and replication, 19 is cyclophilin-dependent. However, in a recently reported highly sensitive NS2 protease assay 3 we did not detect any inhibition of proteolytic activity in the presence of CsA (data not shown). This could be due to a different, yet unknown function of uncleaved NS2- NS3 that is targeted by CsA or the NS2 protease activity is CsA-sensitive only in the context of a replicating virus, but not when assayed in the above-mentioned virus-free protease assay. In fact, a possible molecular mechanism of CsA action against NS2 is suggested by the NS2 crystal structure that revealed an unusual cisproline in position 164 of NS2 that is fully conserved among all HCV-isolates. 20 The peptidyl-prolyl cistrans isomerase activity of CyPs catalyzes the isomerization of peptide bonds from the trans form to the cis form. As Pro 164 lies in close proximity to the protease active site where His 143, Glu 163, and Cys 184 form the catalytic triad, CyP inhibition may preclude correct folding of the NS2 protein backbone and active site formation. In fact, when mutating prolin 164, which is conserved among all known HCV isolates to glycine or alanine, both mutations heavily impaired HCV RNA replication to background levels of the luciferase replication assay (Supporting Fig. 1). In summary, we highlight a new function of CypA as a host factor for HCV and an additional target of action for the CsA-derived antivirals that are currently in clinical trials. The anti-ns2 activity of CsA provides a rationale to test combinations of CsA-derivatives with established agents and novel antivirals targeting other components of the HCV replication machinery. Future clinical studies should address the in vivo benefits of such combination therapies and future laboratory work will be needed to determine which function of NS2 is inhibited by CsA. Acknowledgment: We thank Takaji Wakita and Jens Bukh for JFH1 and J6CF isolates, respectively, and Charles Rice for Huh-7.5 cells. We thank Novartis (Basel, Switzerland) and Debiopharm (Lausanne, Switzerland) for providing reagents. We also thank Jeremy Luban and Thomas Pertel for provision of the CypA and CypB knockdown cell lines. References 1. Simmonds P, Bukh J, Combet C, Deleage G, Enomoto N, Feinstone S, et al. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. HEPATOLOGY 2005;42: Lohmann V, Korner F, Koch J, Herian U, Theilmann L, Bartenschlager R. 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