Multiple Cyclophilins Involved in Different Cellular Pathways Mediate HCV Replication☆

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Virology 397 (2010) 43–55

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Multiple cyclophilins involved in different cellular pathways mediate HCV replication☆

L. Alex Gaither a, Jason Borawski a, Leah J. Anderson a, Kara A. Balabanis a, Piroska Devay a, Gerard Joberty b, Christina Rau b, Markus Schirle a, Tewis Bouwmeester a, Craig Mickanin a, ShanChuan Zhao a, Chad Vickers a, Lac Lee a, Gejing Deng a, Jeremy Baryza a, Roger A. Fujimoto a, Kai Lin a, Teresa Compton a, Brigitte Wiedmann a,⁎

a Novartis Institutes of Biomedical Research, 250 Massachusetts Ave., Cambridge, MA 02139, USA b Cellzome AG, Meyerhofstrasse 1, D-69117 Heidelberg, Germany

article info abstract

Article history: Three cyclophilin inhibitors (DEBIO-025, SCY635, and NIM811) are currently in clinical trials for hepatitis C Received 30 July 2009 therapy. The mechanism of action of these, however, is not completely understood. There are at least 16 Returned to author for revision cyclophilins expressed in human cells which are involved in a diverse set of cellular processes. Large-scale 25 August 2009 siRNA experiments, chemoproteomic assays with cyclophilin binding compounds, and mRNA profiling of Accepted 28 October 2009 HCV replicon containing cells were used to identify the cyclophilins that are instrumental to HCV replication. Available online 24 November 2009 The previously reported cyclophilin A was confirmed and additional cyclophilin containing pathways were fi Keywords: identi ed. Together, the experiments provide strong evidence that NIM811 reduces viral replication by HCV inhibition of multiple cyclophilins and pathways with protein trafficking as the most strongly and Cyclophilin persistently affected pathway. Cyclosporin A © 2009 Elsevier Inc. All rights reserved. NIM811

Introduction proteins to complete its life cycle (examples in (He et al., 2007), (Randall et al., 2007), and (Polyak, 2003)). Targeting host proteins More than 170 million people worldwide are chronically infected represent a successful anti-HCV strategy as proven by the viral load with the hepatitis C virus (HCV), a major cause of chronic hepatitis, reduction in the clinical trial of the cyclophilin inhibitor DEBIO-025 cirrhosis, and hepatocellular carcinoma (World Health Organization, (Flisiak et al., 2008). 1999). Chronic HCV infection has also become the most common Cyclosporin A (CsA) has been shown to have anti-HCV activity indication for liver transplantation in the US. The current therapy in combination with interferon in a clinical trial (Inoue et al., 2003). includes pegylated interferon and ribavirin (Feld and Hoofnagle, Recently, a 3.5 log reduction of viral load has been demonstrated 2005). Unfortunately, this treatment covers only about 50% of the with the CsA analog DEBIO-025 (Flisiak et al., 2008). The prevalent genotypes and is poorly tolerated (reviewed in (Feld and compounds are inhibitors of cyclophilins, proteins of the peptidyl- Hoofnagle, 2005)). Thus, new drugs with broad genotype activity and prolyl cis-trans isomerase family. In humans, nineteen cyclophilin low toxicity are in high demand. isoforms with different cellular and tissue distribution have been HCV is a small enveloped virus that belongs to the Hepacivirus described (Galat, 2004). They are all potential targets of CsA and its genus of the Flaviviridae family. Its RNA genome encodes a single analogs such as DEBIO-025 and NIM811, but only a few cyclophilins polyprotein precursor of about 3,000 amino acid residues, which is have been studied in more detail. The knowledge on the cellular cleaved by cellular and viral proteases to yield at least 10 viral functions of the various cyclophilin isoforms and how they proteins (reviewed in (Branch et al., 2005)). These proteins are modulate HCV replication is currently limited. necessary for the replication of viral RNA and for the assembly of Several laboratories investigated the roles of individual cyclo- viral particles. In addition, the virus depends on numerous host philins in HCV replication and their results have been inconsistent. Employing the replicon in vitro system, cyclophilin B but not A was shown to be involved in viral RNA replication (Watashi et al., ☆ The involvement of Cyp40 in HCV replication was published while the manuscript 2005). Nakagawa et al (Nakagawa et al., 2005) showed that siRNA- was being reviewed by Goto, Watashi et al. (Goto, Watashi et al., 2009, Cancer Science mediated reduction of mRNA levels of cyclophilin A, B, or C 100, 1943-1950. decreases replicase activity while three laboratories reported ⁎ Corresponding author. Infectious Diseases, Novartis Institutes for Biomedical Research, 500 Technology Square, Cambridge, MA 02139, USA. Fax: +1 617 871 5867. cyclophilin A as the critical host factor for HCV (Yang et al., E-mail address: [email protected] (B. Wiedmann). 2008; Chatterji et al., 2009; Hanoulle et al., 2009). We analyzed the

0042-6822/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2009.10.043 44 L.A. Gaither et al. / Virology 397 (2010) 43–55 influence of multiple cyclophilins in the replicon system using not important for this specific replicon model. Cyclophilin G RNAi, pharmacological tool compounds, and quantitative chemical depletion even stimulated replication. proteomics to demonstrate that the replicon depends on the activity of several cyclophilins. We performed siRNA screens and Identification of proteins interacting with NIM811 and CsA using compound competition experiments in HCV replicon cells to detect chemical proteomics proteins in pathways where cyclophilins were involved. In addition, we employed the cyclophilin-binding compounds, NIM811 and CsA, To identify cellular and viral proteins from Clone A cells interacting in RNA profiling studies to identify pathways affected in naïve vs. specifically with NIM811 and CsA but not with the inactive compound replicon-containing cells. Together, these approaches demonstrate PSC833, we performed an affinity purification experiment using an that HCV replication relies on several cellular pathways linked to active analog of NIM811 (NIM811-NH2) linked to Sepharose beads cyclophilins A, H, 40, and E. as a matrix. Clone A cell lysate was preincubated for 30 min with competing compounds cyclosporin A (CsA) and NIM811, the non- Results competing cyclosporin analog PSC833, or DMSO as control. NIM811-

NH2 beads were then added for 30 min and proteins bound to the Cyclophilin binding correlates with activity in replicon assay matrix were purified. Protein targets of active cyclosporin analogs are expected to bind (directly or via a bridging protein that could be We have demonstrated previously that the activity of cyclosporine cyclophilin) free CsA and/or NIM811 and be poorly retrieved by the derivatives in an HCV replicon assay correlates with the binding to NIM811-NH2 matrix in presence of these drugs, but be fully cyclophilin A (Ma et al., 2006). We extended the cyclophilins analyzed precipitated when only DMSO or the inactive compound PSC833 to include Cyclophilin B, C, F, 40 and H (Table 1) and used Surface was added. All four purifications (in presence or absence of competing plasmon resonance (SPR) to study the interaction of CsA and NIM811 compounds) were performed in parallel and eluted proteins were with cyclophilins (Table 2). SPR has the advantage to operate with separated by SDS-PAGE. Samples were processed, labeled with iTRAQ unmodified compounds and provides information on kinetic para- and analyzed by LC-MS/MS for protein identification and quantifica- meters. With both methods we obtained similar results: NIM811 has a tion as described in Material and methods. Supplementary table S2 higher affinity for all cyclophilins than CsA and the binding affinity lists all 636 proteins that were identified by MS. Out of these, 327 met ranks in the order Bb A/Fb Cb Hb 40. The higher affinity of NIM811 the acceptance criterion for robust quantification of four or more vs. CsA is due to a larger kon and smaller koff for all cyclophilins spectra matching to unique peptides. As expected, few proteins were resulting in a lower KD. strongly competed by 5 μM of PSC833. In contrast, a large number of We also tested Sanglifehrin A derivatives, cyclophilin-binding proteins were strongly competed in presence of 5 μM NIM811 or CsA. macrocycles of different chemical structure (Sedrani et al., 2003) and Table 3 lists 51 proteins that were competed at least 50% with both investigated their binding activities to cyclophilins and their HCV NIM811 and CsA but less than 25% with PSC833. Not surprisingly, inhibitory activities. Several derivatives bind to cyclophilins A, B, C amongst the most strongly competed proteins by CsA and NIM811 but and F and inhibit HCV replication (Table 1). The sanglifehrin A analog, not PSC833 are several cyclophilins. Out of nine quantified cyclophi- compound 7 like the cyclosporin analog PSC833 binds much weaker lins, seven were competed more than 85% in presence of both NIM811 to cyclophilins and exhibits low activity in the HCV replicon assay. and CsA (PPIH, PPIA, PPIL1, PPIE, PPIF, PPIB, PPIC). Other highly Only cyclophilin-binding cyclosporin or sanglifehrin analogs reduced competed proteins include splicing factors (SNRPs, PRPFs, LSMs) and viral replication suggesting that one or more of these cyclophilins play extracellular matrix proteins (Laminins, Syndecans, Leprecans). HCV a role in HCV replicon maintenance. non-structural protein NS4B was precipitated by the matrix but the identification was not strong enough to allow for robust quantifica- siRNA-mediated down-regulation of mRNA levels of cyclophilins inhibits tion. Competed proteins can be grouped according to their subcellular viral replication localization and function (Fig. 2). They could possibly associate with the cyclophilins present in the same compartment. Two large clusters The binding affinities of the compounds to the different represent proteins involved in mRNA splicing and ER localized cyclophilins varied up to ten-fold (Tables 1 and 2). Since the proteins. The latter group includes extracellular matrix proteins en cellular cyclophilin levels vary by the same factor (GeneCard®, route to the plasma membrane and their secretion. XenneX, Inc.), it was not possible to identify a particular cyclophilin as the crucial mediator for replication of viral RNA by siRNA-mediated down-regulation of mRNAs of the proteins pulled-down correlating binding affinities and effect on replication. To directly by NIM811 and CsA reveals impact on viral replication investigate the roles of the different cyclophilins, we down- regulated the mRNA levels of individual cyclophilins using siRNAs The identified hits from the pull-down experiments were tested and measured the effect on viral RNA replication by monitoring for their requirement in viral RNA replication. Multiple siRNAs activity of the reporter gene firefly luciferase or by quantifying viral targeting each the mRNA of the proteins listed in Table 3 were RNA levels by QRT-PCR. Fig. 1a shows that each siRNA used transfected into Huh-Luc/neo-ET cells and the effect of the specifically reduces its target mRNA when compared with a non- reduction of the mRNA levels was analyzed by measuring luciferase targeting siRNA, with small effects on expression of the other reporter gene activity. In order to reduce the siRNA number, only cyclophilins. Cyclophilin E siRNA results also in a slight (b25%) one or two of several hits were included in this validation process decrease of cyclophilins A, B, and C. Down-regulation by siRNA of when these hits are known to form complexes such as the some cyclophilins caused an upregulation of others, e.g. cyclophilin spliceosomal complex. Table 4 shows the siRNAs which reduce A depletion resulted in a higher expression of cyclophilins B, C, and viral replication ≥40% as compared to ‘no siRNA’ control. Several 40. In Huh-Luc/neo-ET cells, siRNA mediated knockdown of siRNAs targeting LEPREL1, LEPRE1, PPWD1, HSPA8, RAB43, AGRN, cyclophilin A has the largest impact on replication, with less than SART3 reduced viral replication. One or two siRNAs were identified 20% of replicon activity remaining after 96 h (Fig. 2b). Cyclophilin to reduce viral replication for the spliceosomal proteins SNRPD1 H, 40, and E knockdown had a less pronounced but statistically and 3, SNRPB, SNRPN, NHP2L1, XAB2, SNW1. Although none of the significant effect (p=0.04, 0.03 and 0.02 at 96 h, respectively, siRNAs targeting LSM3 and 4 had an effect on replicon activity, compared with non-targeting siRNA by students t-test), decreasing siRNAs targeting components of the same complex, LSM 2 and 6, replicon activity by 30–50%, whereas cyclophilins B, C, and F are were able to reduce viral replication. L.A. Gaither et al. / Virology 397 (2010) 43–55 45

Table 1 Correlation of anti-HCV and cyclophilin-binding activities of compounds.

Compound Replicon Cyp A Cyp B Cyp C Cyp F Cyp 40 Cyp H

IC50 [μM] KD [nM] CsA derivatives CsA 0.45 26 14 190 30 360 250 PSC833 12 2600 3400 N3500 2100 N3300 N3300 NIM811 0.10 13 3.4 32 6.7 130 82 NIM811-NH-Boc 0.15 23 6.9 67 13 220 92

Sanglifehrins Compound 3 2.5 19.7 10.3 31.9 50.0 496.7 78.7 Compound 4 4.11 223.0 79.3 425.5 800.0 2516.6 160.7 Compound 5 5.6 19.7 6.9 35.5 60.0 993.3 88.5 Sanglifehrin A 5.9 13.8 21.4 9.2 Compound 6 200 N3278.7 N3448.3 N3546.1 N3333.3 N3311.3 N3278.7 Compound 7 0.13 14.1 9.3 13.5 21.7 185.4 28.5

Compound 4 was previously published with an IC50 of 30 nM for CypA (Sedrani, Kallen et al., 2003).

Screening of siRNA libraries revealed cellular proteins involved in protein further validated with four distinct siRNAs targeting each gene and trafﬁcking as important for viral replication only those where two or more siRNAs reduced viral replication were considered as hits. Fig. 3 summarizes the identiﬁed proteins Next we screened two siRNA libraries in Huh-Luc/neo-ET cells of both screens clustered into pathways. The detection of pathways to identify cellular proteins and pathways necessary for replication. known to affect HCV replication such as pyrimidine-de-novo Two libraries were tested: a kinome siRNA library and a library synthesis and cholesterol biosynthesis validated the screen. Con- containing siRNAs targeting ∼3000 druggable genes. The hits were sistent with the data obtained by our other approaches, the largest 46 L.A. Gaither et al. / Virology 397 (2010) 43–55

Table 2 Kinetics and binding afﬁnity of CsA and NIM811 to cyclophilins.

Cyclophilin A Cyclophilin B Cyclophilin C Cyclophilin H Cyclophilin 40

NIM811 CsA NIM811 CsA NIM811 CsA NIM811 CsA NIM811 CsA

− 1 − 1 5 5 5 5 5 5 5 5 5 5 kon (M s )8×10 4×10 7×10 4×10 6×10 2×10 2×10 1×10 5×10 2×10 − 1 − 1 koff (M s ) 0.005 0.010 0.002 0.003 0.006 0.010 0.008 0.020 0.030 0.080

t1/2 (min) 2.30 1.18 7.45 3.73 1.81 1.00 1.46 0.67 0.37 0.14

KD (nM) kinetic 6.14 24.7 2.23 7.96 10.8 52.6 32.5 142 58.0 441

number of hits fell into protein/lipid trafficking and spliceosome centration and time dependent with a larger effect at higher concen- pathways. tration and shorter treatment time (data not shown). The affected mRNAs were overlapping in each treatment: the same pathways were Comparison of mRNA profiles of naïve versus Clone A cells with either down- or up-regulated independent from drug concentration and without NIM811 or CsA treatment or treatment time. NIM811 and CsA caused similar changes at the level of gene expression but NIM811 was more potent at comparable We performed two mRNA profiling experiments with NIM811 and concentration and treatment time. CsA in naïve Huh7 cells and Clone A replicon cells (Tables 5, 6, com- The Gene Set Enrichment Analysis (GSEA) detects small and plete data set is available http://www.ncbi.nlm.nih.gov/geo). The coordinated changes of a functionally related group of genes from mRNA changes observed after compound treatment were both con- expression analysis data (Szustakowski et al., 2006). GSEA takes in

Fig. 1. siRNA-mediated reduction of mRNA levels of four cyclophilins causes decrease in HCV replication. (a) 25 nM siRNA targeting cyclophilins or a non-targeting siRNA were transfected into Huh-Luc/neo-ET cells and the mRNA levels of their targets and other cyclophilins quantiﬁed relative to a non-targeting siRNA after 72 h (gene expression for the non-targeting siRNA was set to a value of 1, not shown). (b) Replication was measured by luciferase activity at 72 and 96 h after siRNA transfection. Relative replicon activity for each siRNA is shown compared with a non-targeting siRNA. CypA, cyp40, cypE and cypH siRNAs result in a statistically signiﬁcant decrease in viral replication at both time points (pb0.05 by Student's t-test compared with non-targeting siRNA). L.A. Gaither et al. / Virology 397 (2010) 43–55 47

Fig. 2. Distribution of NIM811 and CsA binding proteins to cellular compartments and pathways. Proteins interacting with NIM811 and CsA but not with the control substance PSC833 (see Table 3) as determined by in-lysate competition are clustered to their location and possible interaction with cyclophilins. Reduction of mRNA levels of the bold labeled proteins by siRNA reduced viral replication at least 40% (Table 4). siRNA of the proteins in brackets and bold font have not been tested with siRNA but siRNAs targeting other components of the complex these proteins are integrated in were identified. Proteins in brackets did not score in the siRNA test but they are known components of a protein complex for which other members showed influence on HCV replication. account the number of regulated genes per pathway and the al., 2007; Chatterji et al., 2009; Hanoulle et al., 2009). Chemically strength of regulation and expresses both as a significance value (p- unrelated cyclophilin inhibitors, the sanglifehrins, exhibit the same value). Since genes can be part of several different pathways, we effect supporting the hypothesis that (a) cyclophilin(s) is/are performed a clustering of pathways by combining pathways which necessary for HCV replication. Nineteen open reading frames encod- contain the same regulated genes into one cluster. Table 5 ing cyclophilins have been identified in the human genome. All summarizes the pathways most significantly regulated by the contain a conserved catalytic domain that is responsible for the cis/ replicon grouped into pathway clusters. In addition, the table trans isomerisation of peptidyl-prolyl amino acid bonds (Galat, 2004). contains information on how these pathways were regulated in the Cyclophilins are distinct in their tissue expression and subcellular presence of NIM811 and how they are affected by NIM811 in naïve localization. We sought to identify host factors or pathways that cells. In replicon containing cells, genes involved in protein regulate viral replication using small interfering RNA-mediated biosynthesis were down-regulated while genes in immunity and down-regulation of mRNAs, compound pull-down, and mRNA ATP generating pathways were up-regulated. For most pathways, profiling. Together, the results of these approaches lead to the treatment of replicon-containing cells with NIM811 induced conclusion that several cyclophilins are necessary for HCV replication. changes that are inversely correlated to the changes induced by A similar observation was reported recently (K.-I. Abe et al., abstract the replicon itself. This inverse relationship is most likely due to 387, presented at the 15th International Symposium on Hepatitis C inhibition of viral replication since NIM811 has no effect in naïve Virus and Related Viruses, San Antonio, TX, 2008). Our mRNA Huh7 cells (Table 5, column 5). One exception is intracellular depletion experiments showed that cyclophilin A, H, and to a lesser protein trafficking, which is strongly up-regulated in naïve cells by extent 40, and E participate in pathways required for efficient viral NIM811 treatment (Table 6) as well as by the replicon. Additionally, replication in Huh-Luc/neo-ET cells. cadherin signaling and developmental processes were down- The results of the siRNA experiments are supported by the pull- regulated by NIM811 and by the replicon (Tables 5 and 6). Table down of proteins in pathways containing different cyclophilins. The 6 demonstrates that protein trafficking was the most significantly spliceosome contains several cyclophilins, such as cyclophilin H, E, L1, up-regulated pathway by NIM811 treatment. Processes that depend L3 and PPWD1. Therefore it was not surprising that a large number of on the transport of proteins to the cell surface and mediate cell–cell proteins (∼37% of all hits) interacting with these cyclophilins were contact, cell adhesion and communication, were down-regulated. identified in the compound pull-down experiment even though the affinity of the compounds to CypH is weaker than to other cyclophilins Discussion (Table 1). The observation that a decrease of spliceosomal proteins by siRNA treatment diminished viral replication without being toxic to The anti-HCV activity of cyclosporin A and NIM811 has been the cell was quite unexpected since the viral RNA does not need attributed to their binding to cyclophilin B (Watashi et al., 2005; Liu et splicing. The largest group of proteins (∼43%) identified in the pull- al., 2009) and recently to cyclophilin A (Yang et al., 2008; Fernandes et down experiment is involved in protein trafficking processes and 48 L.A. Gaither et al. / Virology 397 (2010) 43–55

Table 3 Table 4 Proteins showing significantly stronger competition by NIM811 and CsA (N50%) over NIM811/CsA interacting proteins affecting viral replication. PSC833 (b25%). Accession no. Average RLU Number of siRNAs GeneData ID Representative Gene Competition relative mRNA mRNA NM_198576 2912 5 AGRN to DMSO control regulated regulated NM_052857 2860 1 CCDC16 in replicon in NIM CsA NIM811 PSC833 NM_006597 2810 6 HSPA8 containing treated vs. NM_005561 2697 6 LAMP1 vs. naive untreated NM_022356 2857 6 LEPRE1 Huh7 cells Huh7 cells NM_018192 2890 4 LEPREL1 IPI00479925.1 AGRN 89.4 90.7 22.1 NM_021177 3226 3 LSM2 IPI00297572.4 AQR 81.7 84.9 7.3 NM_007080 3020 1 LSM6 IPI00420014.2 ASCC3L1 57.2 62.1 −29.5 NM_001003796 2992 5 NHP2L1 IPI00440493.2 ATP5A1 69.8 53.3 −8.7 NM_015342 3104 5 PPWD1 IPI00043565.3 CCDC16 79.3 76.0 −89.2 1.7 NM_004697 3240 2 PRPF4 IPI00437593.1 COL18A1 79.1 78.3 9.8 −1.7 NM_020701 3240 4 RAB43 IPI00220959.7 CRTAP 88.7 77.1 −144.2 1.7 NM_138421 3147 3 SAAL1 IPI00177366.2 DHX15 70.8 64.7 −45.2 NM_014706 3115 4 SART3 IPI00001755.1 GPC6 85.5 75.1 −15.5 NM_198216 3220 1 SNRPB IPI00031522.2 HADHA 87.3 86.6 −7.9 NM_006938 2720 1 SNRPD1 IPI00003865.1 HSPA8 73.4 55.9 −86.9 NM_004175 3273 3 SNRPD3 IPI00024284.4 HSPG2 81.5 76.6 11.8 NM_003095 2980 1 SNRPN IPI00641693.2 LAMA5 90.6 88.8 11.9 NM_012245 2980 1 SNW1 IPI00013976.1 LAMB1 88.4 84.8 −5.4 NM_020196 2960 1 XAB2 IPI00004503.4 LAMP1 75.4 64.2 −9.5 1.7 888 (n=18) pGL2 IPI00045839.2 LEPRE1 96.9 92.8 −1.8 5658 (n=22) no siRNA IPI00217055.1 LEPREL1 89.9 83.4 −7.6 Multiple siRNAs per gene for the proteins identified as NIM811/CsA interacting in pull- IPI00032460.3 LSM2 80.8 90.2 3.8 down competition assays were tested at 25 μM concentration in cells containing the IPI00219229.2 LSM3 78.9 85.2 5.7 Huh-Luc/neo-ET replicon. The luciferase reporter signal was measured in duplicate IPI00001146.1 LSM6 77.0 78.5 −4.9 plates. Proteins for which the average luciferase signal was decreased ≥40% compared IPI00007163.1 LSM7 74.5 83.3 −6.7 to the ‘no siRNA’ signal are listed in the table. IPI00219871.4 LSM8 82.2 86.4 19.5 IPI00026167.2 NHP2L1 84.1 88.3 14.0 IPI00026944.2 NID1 73.9 68.6 −10.3 −1.9 In the following section we will link the evidence from our IPI00480133.1 PPIA 95.6 96.2 −7.2 experiments for the function of various cyclophilins with their roles IPI00024129.1 PPIC 92.3 88.7 10.9 described in the scientific literature. This analysis suggests that several IPI00003927.4 PPID 66.1 77.9 14.1 −1.5 IPI00514173.1 PPIE 93.3 91.2 −0.6 cyclophilin-containing pathways are necessary for viral replication. IPI00026519.1 PPIF 95.6 90.5 −18.4 IPI00007346.1 PPIH 92.5 97.4 −38.5 IPI00007019.1 PPIL1 89.3 93.9 −11.3 IPI00149650.3 PPWD1 66.4 84.1 9.2 IPI00021146.1 PRPF18 67.3 83.9 −0.1 1.6 −2 IPI00005861.1 PRPF3 78.8 85.2 3.5 IPI00292000.6 PRPF31 78.3 77.4 −50.9 IPI00150269.1 PRPF4 77.4 84.0 −14.5 1.7 IPI00007928.4 PRPF8 66.9 66.1 11.1 IPI00556031.2 RAB43 84.5 82.2 −23.4 IPI00304935.4 SAAL1 80.6 73.0 −17.7 IPI00006025.1 SART3 70.7 80.4 5.9 IPI00002441.1 SDC1 93.2 91.6 17.5 −1.7 IPI00300407.3 SDC2 94.4 93.7 10.4 1.5 IPI00329512.3 SNRPB 69.7 75.9 −4.8 IPI00017963.1 SNRPD2 71.8 78.9 18.3 IPI00029266.1 SNRPE 77.7 82.8 0.0 −2.5 IPI00220528.6 SNRPF 66.4 64.0 −10.3 IPI00016572.1 SNRPG 74.0 80.4 −21.1 IPI00013830.1 SNW1 84.0 81.9 −10.3 1.5 IPI00290566.1 TCP1 76.4 57.8 −62.4 IPI00163084.3 XAB2 90.7 91.0 −12.3 −2.2

The mRNA levels are presented as fold-shifts and 1.5 was used as the cut-off value.

appears to interact with cyclophilins B and C. HCV non-structural proteins NS3, NS4B, NS5A and NS5B were precipitated by the matrix but none have been significantly competed by the addition of either NIM811 or CsA, indicating that the possible interaction between viral proteins and cyclophilins is weaker than the interaction of cyclophilins with known cellular interacting proteins. Alternatively, the interaction may be of a transient nature, and conformational changes upon binding may occur which prevent a stable interaction. The NS5A and NS5B proteins have recently been shown to be substrates of Fig. 3. Host proteins identified in siRNA screens to affect HCV replication. The kinome cyclophilins A and B (Hanoulle et al., 2009). The same pathway, and the 5000 druggable gene siRNA libraries were transfected into Huh7 cells containing the Huh-Luc/neo-ET replicon and their effect was measured by luciferase protein trafficking, has been identified to be affected by the viral reporter gene activity. Hits from the two screens were further tested by a secondary fi replicon as well as by NIM811 with a gene expression pro ling siRNA knock-down experiment. Proteins identified in both experiments were clustered approach. into cellular pathways. L.A. Gaither et al. / Virology 397 (2010) 43–55 49

Table 5 Modulation of pathways by HCV replicon and/or NIM811 treatment.

Pathway cluster Pathway name Replicon vs. NIM811 treated NIM811 treated Huh-7 cells vs. untreated vs. untreated Replicon cells Huh-7 cells

Downregulated Protein biosynthesis Ribosome −7.7 1.0 −0.1 Protein biosynthesis −4.5 0.4 2.5 Aminoacyl-tRNA biosynthesis −6.6 0.4 0.4 Map00970_aminoacyl_tRNA_biosynthesis_mootha −5.9 0.2 0.2 Amino acid activation −5.8 0.4 0.4 Other Developmental processes Cadherin signaling pathway −3.9 −0.7 −8.5 Oncogene −3.6 0.8 −0.7 Glycosylphosphatidylinositol(gpi)-anchor biosynthesis −3.6 0.7 −0.7 Ectoderm development −3.1 −0.4 −3.8 Other apoptosis −3.0 1.6 −0.3

Upregulated Other Intracellular protein trafﬁc 5.0 5.8 61.0 Sulfur metabolism 5.2 −1.2 1.8 Mitosis 5.7 −0.7 0.8 Cytokinesis 5.7 −0.8 −0.2 Map03070_typt_III_secretion_system_mootha 5.7 −1.9 1.2 Signaling signaling:homosapiens:disease:rheumatoid arthritis:osm 5.3 −1.1 −0.2 signaling pathway signaling:homo sapiens:disease:acquired immuno deﬁciency 5.8 −1.3 1.9 syndrome:tat signaling pathway signaling:homosapiens:physiology:cell adhesion:csf signaling pathway 7.8 −1.5 −0.3 Energy generation Glycolysis/gluconeogenesis 5.3 −7.5 0.5 Map00193_atp_synthesis_mootha_human 5.7 −1.9 1.2 Map00195_photosynthesis_mootha_human 6.0 −2.0 1.1 Mitochondria_mootha_human 8.0 −15.3 0.1 Atp synthesis 8.4 −7.5 0.4 Oxidative phosphorylation 9.0 −21.1 −0.1 Human_mitodb_6_2002_mootha_human 9.4 −18.3 −0.2 Carbohydrate metabolism 11.4 −6.2 1.4 Immunity t-cell mediated immunity 5.5 −4.5 −1.9 Mhc I-mediated immunity 11.6 −4.9 −1.0

The numbers represent the negative exponent of the p value reported in GSEA. The p value expresses the probability that a gene set is enriched by chance. The “−” preceding the number indicates the directionality of regulation; e.g. if it is negative it indicates an overall down-regulation, if it is missing it indicates an up-regulation. The pathways were sorted to identify the strongest regulation in “replicon vs. Huh-7 cells.”

Cyclophilin A oxysterol-binding protein which is involved in the phosphatidylinositol-regulated homeostasis of cholesterol via regulation of HMG-CoA Cyclophilin A is reported to be a cytosolic soluble enzyme and its reductase. hVAP-A mediates vesicle transport together with the most commonly known function is the modulation of T-cell mediated phosphatidylinositol-transfer protein Sec14 (Wyles and Ridgway, immune response in a complex with cyclosporin A. The cyclosporin- 2003). The required PI(4)P is generated by PIK4Cs identified in our cyclophilin complex binds to calcineurin and inhibits its phosphatase siRNA screen to be essential for HCV. PI4KA has been demonstrated by activity which prevents the migration of NF-κB into the nucleus where several other laboratories to be involved in viral replication (Berger et it activates gene transcription of IL-2, IFN and other chemokines al., 2009; Tai et al., 2009; Vaillancourt et al., 2009). These results typical for the T-cell immune response (Clipstone and Crabtree, indicate that HCV depends on an intact protein and lipid transport 1992). This mechanism is exploited when CsA is used to prevent pathway. The mRNA profiling experiment showed that expression of rejection of transplants by inflammatory responses (Borel, 2002). the replicon up-regulates sterol as well as phosphatidylinositol Besides its role as cis/trans prolyl isomerase cyclophilin A has also biosynthesis. Inhibition of sterol biosynthesis by statins has been been implicated in cholesterol transport. It has been detected in demonstrated to cause reduction of viral replication (Ikeda and Kato, complex with FKBP56, cyclophilin 40 and caveolin transporting 2007). siRNAs targeting mRNAs in the sterol biosynthesis pathway cholesterol in a CsA sensitive manner to caveolae (Uittenbogaard et (MVD) or in the trafficking pathway (PIK4C) decreased replication. al., 1998). Another mechanism of cytosolic cholesterol transport Cyclophilin A might contribute to the highly sensitive cholesterol between different membranes is mediated by lipid droplets which homeostasis by mediating its transport. HCV depends on cholesterol also contain caveolin and, as recently explored to greater detail, HCV and any disturbance in cholesterol level or distribution will decrease proteins (Boulant et al., 2007;Miyanari et al., 2007). Overexpression of replication. Rab18 causes disappearance of adipophilin from lipid droplets and A third function of Cyclophilin A, regulating transcription, has been subsequently to the association of the droplets with the ER next to the demonstrated recently. Cyclophilin A interacts with Sin3A/Rdp3 translocon, the site where the HCV core protein enters the membrane HDAC complexes and modulates HDAC-dependent gene silencing in and the droplets bud (Ozeki et al., 2005). Lipid rafts are yet another yeast (Shaw, 2002). The ortholog complex regulates transcription of vehicle for protein and lipid transport. These membrane domains are the MYC target gene CAD in humans (Pal et al., 2003). CAD, an enzyme highly enriched in cholesterol and sphingolipids and have been in the pyrimidine de-novo biosynthesis, was one of the strongest hits shown to be the site of HCV replication (Gao et al., 2004; Shi et al., in our siRNA screen and it is known that HCV depends on the 2003). hVAP proteins, residents of lipid raft, interact with NS5A and pyrimidine de-novo biosynthesis pathway (Stuyver et al., 2003). NS5B and mediate the formation of the replicase complex (Gao et al., In humans, antiviral responses depend on the activation of 2004; Hamamoto et al., 2005). hVAP-A also interacts with an transcription, a process that requires the chromatin-remodeling 50 L.A. Gaither et al. / Virology 397 (2010) 43–55

Table 6 and stimulates T-lymphocytes (Allain et al., 2002). Proteoglycan core Pathways which are most highly regulated by treatment with NIM811 in naïve Huh7 protein and several proteoglycans have been pulled-down in our cells. assay together with proteins of the transport machinery (Rab43, Pathway Pathway name NIM811 treated vs. RRBP1, and LEPREL1). Smith et al. (1995) demonstrated that cluster untreated Huh7 cells cyclophilin B transports procollagen from the ER to the Golgi. Downregulated Transport of ApoB to the Golgi had been shown to occur in complex Cell–cell Cell adhesion −12.5 with cyclophilin B and ER chaperones (Zhang and Herscovitz, 2003). It − contact Cell-adhesion mediated signaling 12.3 is therefore possible that cyclophilin B not only binds surface proteins Cell communication −12.2 Cadherin signaling pathway −8.5 in the extracellular space, but that it interacts with these proteins and Developmental processes −8.3 others such as viral proteins which travel via the secretory pathway as Wnt pathway −7.6 soon as they are synthesized and enter the ER through the translocon Extracellular matrix protein-mediated −7.6 pore. Cyclophilin B has to be in close proximity of the translocon since signaling it has been crosslinked to Sec61 (Smith et al., 1995). Protein Cell structure and motility −6.8 fi fi Cell structure −6.4 traf cking was the most signi cantly affected pathway by treatment Other C24_mootha_human −12.2 with NIM811 and CsA. It was also up-regulated in Clone A replicon- Signal transduction −11.4 containing cells compared to naïve cells indicating that this replicon − Chromatin packaging and remodeling 8.9 depends on the protein trafficking pathway. siRNAs leading to − Signaling:homo sapiens:disease: 5.6 fi glioblastoma:responsive genes reduced levels of proteins involved in protein traf cking and Coagulation Complement and coagulation cascades −5.4 maturation, two processes that depend on each other, reduced viral Blood clotting −4.0 replication. Recently, glycosyltransferases have been suggested as anti-viral targets (Dwek et al., 2002). UDP-glycosyltransferase has Upregulated been isolated in a large ER protein complex bound to nascent proteins Other Signaling:reference:physiology:apoptosis: 3.4 fi trail mediated apoptosis which also contained protein disul de isomerase (PDI), cyclophilin B Signaling:homo sapiens:disease:prostate and other proteins (Meunier et al., 2002). We found proteins involved cancer:trail signaling pathway in the folding/maturation of proteins of the secretory pathway to be Signaling:homo sapiens:disease:melanoma: 3.5 necessary for viral replication (e.g. LEPRE1, LEPREL1, RPN1). Leprecan, trail signaling pathway Aminosugar metabolism 3.7 a prolyl-3-hydroxylase modifying collagens among other proteins, C12_mootha_human 8.5 has been observed in complex with cyclophilin B, FKBP65, Hsp47 (a.k. Protein Protein targeting 3.0 a. SERPINH1) and PDI, a prolyl-4-hydroxylase (PDI) (Meunier et al., trafficking Protein disulfide-isomarease reaction 3.2 2002). Leprecan was identified in the compound competition study Protein targeting and localization 3.8 (Table 3) and SERPINH1, PDI3 and 4 were identified in cyclophilin B Constitutive exocytosis 4.7 Protein folding 4.8 pull-down studies (data not shown). Protein export 5.7 Virus amplification and replication has been reported to cause an Exocytosis 8.9 Unfolded Protein Response (UPR) which leads to termination of General vesicle transport 11.3 translation (Tardif et al., 2002). We observed protein biosynthesis Intracellular protein traffic 61.0 down-regulated by the replicon indicating that the replicon can The numbers represent the negative exponent of the p value reported in GSEA. The p induce the same changes as the complete virus. value expresses the probability that a gene set is enriched by chance. The “−” preceding Cyclophilin B has been demonstrated to be essential for virus- the number indicates the directionality of regulation; e.g. if it is negative it indicates an overall down-regulation, if it is missing it indicates an up-regulation. induced interferon beta expression, suggesting indirect involvement of cyclophilin B in virus replication (Obata et al., 2005). A direct interaction SWI/SNF-like BAF complex (Cui et al., 2004). We found that of cyclophilin B with NS5B and NS5A has been demonstrated, an transcription and chromatin structure pathways are regulated by interaction that facilitates RNA binding and replication (Watashi et al., CsA and NIM811 in naïve Huh7 cells—most likely due to inhibiting the 2005; Fernandes et al., 2007). Taken together, there seem to be several interaction of cyclophilin A with HDACs. In an approach to tag ways by which cyclophilin B enables viral replication: by direct cyclophilin A we discovered the protein localizes almost exclusively in interaction with viral proteins and stimulation of RNA synthesis, by the nucleus of Huh7 cells (data not shown). Proteins involved in induction of the UPR, and by influencing protein trafficking. transcription regulation were competed off of cyclophilins by CsA or Down-regulation of cyclophilin B mRNA did not affect replication NIM811 from the NIM811-covered matrix (Table 3). Cyclophilin A of the Huh-Luc/neo-ET replicon in our experiments but we observed a mRNA depletion had a profound effect on viral replication likely due significant small effect on Clone A cells (data not shown). We are to the several functions of this cyclophilin in human cells. Cyclophilin currently exploring whether this is due to the differences in the viral A inhibition can affect viral replication by either interfering with the RNA or protein sequences or a result of experimental conditions. transcription of genes more valuable for the virus than the host cells such as hVAP, La autoantigen or other cellular factors described to be Cyclophilin C necessary for efficient viral replication. Another hypothesis involves modification of virus-induced interferon-mediated gene transcription Cyclophilin C, similar to cyclophilin B, is located in the ER and Golgi which is regulated by HDAC (Cui et al., 2004). Our profiling and can be secreted. Cyclophilin C is induced by interferon (www. experiments revealed a more than 5-fold up-regulation of IL-8 levels lerner.ccf.org/labs/Williams), and is highly expressed in kidney, upon treatment with CsA or NIM811. This chemokine has been shown which is thought to be the reason for the kidney toxicity of CsA to be up-regulated by recruitment of HDAC1 to its promoter in RSV (Friedman et al., 1994). This cyclophilin is involved in the innate infected cells (Jamaluddin et al., 2005). immune response via binding to Mac-2BP (LGALS3BP) (Trahey and Weissman, 1999). Mac-2BP was initially identified as cyclophilin C- Cyclophilin B binding protein (CyC-AP) in mice and the interaction of cyclophilin C with Mac-2BP is disrupted by CsA (Friedman et al., 1993). CyC-AP Cyclophilin B is a membrane bound enzyme of the endoplasmic knock out mice are viable, however their survival is decreased upon reticulum (ER) and Golgi and has also been found to be secreted. endotoxin exposure, which induces IFNγ, TNFα and IL-12 (Trahey and Extracellular cyclophilin B binds to glucosaminoglucans and CD147 Weissman, 1999). CyC-AP is thought to be involved in the host L.A. Gaither et al. / Virology 397 (2010) 43–55 51

Fig. 4. Several cyclophilin-mediated pathways play a role in maintaining HCV. The scheme depicts the HCV life cycle and the main cellular cyclophilins with the pathways they are involved in. These pathways have been identified as important for viral replication in the work presented here. defense system since it is up-regulated both in tumor cells and by Cyclophilin 40 virus infection (HIV, HCV) (Grassadonia et al., 2004). The presence of CyC-AP in body fluids is used as prognostic marker for cancer, We will use the term cyclophilin 40 rather than cyclophilin D for immunodeficiencies and for nonresponsiveness of HCV patients to the cytosolic cyclophilin and cyclophilin F to describe the mitochon- interferon (Artini et al., 1996; D'Ostilio et al., 1996). Besides binding to drial cyclophilin to avoid confusion. cyclophilin C it also interacts with collagens and fibronectin (Sasaki et Cyclophilin 40 is found in the cytosolic complex transporting al., 1998) and it stimulates the expression of MHC class I (Grassadonia cholesterol together with cyclophilin A and FLBP56. It is also a co- et al., 2004). MHC I is one of the most highly regulated pathways in chaperone in the Hsp90/Hsp70 complex which interacts with a replicon-containing cells and can be suppressed by treatment with number of cellular proteins including steroid receptors, kinases such NIM811 (Table 5). Cell adhesion, cell–cell communication, and as Wee, MEK, MAPK, and the transcription factors p53, NF-AT extracellular matrix protein-mediated signaling pathways are (Carrello et al., 2004; Taylor et al., 2001). The biological function of down-regulated by NIM811 and CsA, indicating an involvement of cyclophilin 40 is unknown but it could determine which protein cyclophilin C. Cyclophilin C is the most highly down-regulated interacts with the heat shock protein complex since cyclophilin 40 cyclophilin by the replicon after 48 h and cyclophilin C mRNA binds to Hsp with its TPR domain leaving the CsA binding domain free depletion by siRNA treatment does not decrease viral replication. to trigger conformational changes in client proteins by catalyzing cis- Several proteoglycans which interact with collagen and syndecan trans isomerization of peptidyl-prolyl bonds (Taylor et al., 2001). A have been identified in our pull-down experiments. Based on the direct interaction of Hsp90 with the viral NS2/3 protease has been activities of cyclophilin C and cyclophilin B we hypothesize that they postulated since geldanamycin, an inhibitor of the ATPase function of are involved in the trafficking of viral proteins from their site of Hsp90, blocked the proteolytic function of this viral protease (Wax- translation to the replicase complex. man et al., 2001). Hsp90 has also been reported to directly interact 52 L.A. Gaither et al. / Virology 397 (2010) 43–55 with the NS5A protein of HCV and geldanamycin blocked viral in the spliceosomal apparatus (cyclophilins G and E, PPWD1, PPPIL1, replication (Okamoto et al., 2006). Based on our results, we predict PPIL3 and cyclophilin H). that cyclophilin 40 competes with other TPR domain containing proteins for the binding site of heat shock proteins Hsp90 and/or Cyclophilin H Hsp70 and controls different functions such as protein degradation during the UPR that may be caused by stress induced by expression of As mentioned above, cyclophilin H is another component of the the replicon or virus. eIF-2A kinase is a known substrate of Hsp90 and spliceosome. The PRPF3 and 4 proteins pulled-down with compounds is involved in the translation attenuation during stress caused by are interacting proteins of this cyclophilin. overload of the ER translocation/modification machinery with viral Like many small RNA viruses, HCV is dependent upon the host cell proteins. dsRNA kinase is another example of Hsp90 client proteins and utilizes multiple pathways including protein trafficking, tran- and it is known to be involved in the antiviral host response scription, and splicing for efficient viral replication. The predominant (Balachandran et al., 1998). Thus, there is a large number of Hsp90 cyclophilins (A, B, C, H, F and 40) can be linked to these processes, client proteins known to be involved in antiviral responses which though it is possible that other cyclophilins are involved as well. Fig. 4 could be affected by NIM811 or CsA mediated inhibition of cyclophilin gives an overview of the HCV life cycle and cyclophilins identified in 40. Inhibition of cyclophilin 40 resulted in a moderate decrease of viral our studies with the pathways they are involved in. Although siRNA replication (Fig. 1b). In the pulldown experiment several heat shock screening in combination with drugs successfully identified chemo- proteins were identified with HSPA8 being significantly competed by sensitizer in the past (Whitehurst et al., 2007), the existence of NIM811 and CsA but not PSC833. Pathways including Hsp client multiple targets may explain why it was not possible to identify only proteins are affected by NIM811 and CsA, such as MAP kinases or Fyn one protein by siRNA screening that is involved in synergizing or in signaling pathways or protein folding. antagonizing with NIM811 treatment. Also, it proved extremely difficult to select NIM811-resistant replicons, further supporting the Cyclophilin F possibility of multiple cellular targets. Work will be continued in our laboratory to shed more insight into the relationship of cyclophilins Cyclophilin F is a component of the mitochondrial permeability and the replication of HCV. transition-pore and exhibits anti-apoptotic functions (Schubert and Grimm, 2004). There is controversial evidence as to whether Material and methods cyclosporin treatment provokes pro-apoptotic (Pyrzynska et al., 2002) or anti-apoptotic activities (Nozynski et al., 2007). NIM811 Compounds more strongly upregulates apoptotic pathways than CsA in our profiling experiments, consistent with their binding affinities to cyclophilin F. CsA, NIM811, cyclophilin analogs (PSC833 and NIM811-NH2), NIM811 binds with equal affinity to cyclophilin F, A and B and it is sanglifehrin A, and sanglifehrin derivatives (compounds 3–7) were therefore surprising that the effect on mitochondria is not stronger than synthesized or isolated at Novartis. on protein trafficking, cell–cell contact, or chromatin modeling. Compound treatment induced a complex regulation of energy Cell culture producing pathways in two independent experiments: low concentration of NIM811 up-regulated these pathways whereas at higher The Con1 subgenomic replicon cell line (Clone A) used for concentration it was inhibitory. In a separate time-course experiment transcription profiling and in siRNA experiments was licensed from the same high concentration of NIM811 caused an up-regulation of Apath, LLC (St. Louis, MO). The subgenomic replicon containing a energy producing pathways, especially at an earlier time point (12 vs. luciferase reporter gene (Huh-Luc/neo-ET) employed in siRNA 48 h). The replicon caused up-regulation at all concentrations and experiments and for analysis of compound activities was licensed over time suggesting that the observed drug-mediated regulation is from ReBLikon GmbH, Germany. Huh7 cells were maintained in not an artifact. The number of proteins identified in the pull-down Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen) with 10% experiments that could be associated with cyclophilin F is much Fetal Bovine Serum (Hyclone), 0.1 mM non-essential amino acids smaller than that of cyclophilin B, A or H. In addition, siRNA mediated (NEAA) (Invitrogen), and 2 mM L-glutamine (Invitrogen) at 37 °C knockdown of cyclophilin F expression did not effect viral replication. with 5% CO2 and split 1:4 to 1:8, twice per week. Medium of the Taken together, the role of cyclophilin F in viral replication is limited. replicon-containing cells was supplemented with 250 μg/ml G418 (Invitrogen) for Huh-Luc/neo-ET cells and with 500 μg/ml G418 for Cyclophilin G Clone A cells.

Cyclophilin G (SR-Cyclophilin, Matrin-Cyclophilin) is a nuclear Replicon assay to measure compound inhibitory effects matrix protein which interacts with the phosphorylated C-terminal domain of the largest subunit of RNA polymerase II (POLR2A) The anti-viral activity of compounds was determined using Huh- (Bourquin et al., 1997). It contains a large C-terminal serine/arginine Luc/neo-ET cells. Briefly, 5000 replicon cells were seeded in each well (SR)-rich domain found predominantly in pre-mRNA splicing factors. of a 96-well plate and were allowed to attach overnight without G418 Spliceosomal proteins were the second largest group affecting selection pressure. On the next day, the culture medium was replaced replication identified by siRNA. PPWD1 was identified in our pull- with medium containing serially diluted compounds in the presence down assay together with a large number of splicing factors known to of 10% FBS and 0.5% DMSO. After 48 h compound treatment, the interact with these cyclophilins (AQR, XAB2, LSM2 and 3, CRNKL1, luciferase activities in the cells were determined using the BriteLite PRPF8, SART1, SNRPD1, 2 and 3, SNRPE, SNRPF, SNRPG, SNW1). Viral reagent (Perkin Elmer) with an LMaxII plate reader (Molecular Probe,50 core protein has been demonstrated to interact with alternative Invitrogen). Four replicates were measured for each data point. IC is splicing factor 2 and cause its re-distribution in the cell (A. H. Patel, the concentration of compound at which the luciferase activity in the 14th International Symposium on Hepatitis C virus and related replicon cells is reduced by 50%. viruses, Glasgow, UK, 2007). It is clear that the virus exploits the Cyclophilin binding assay cellular splicing machinery and the disturbance of splicing therefore causes replication to cease. The strong effect of cyclophilin inhibiting Initially, the binding of cyclosporinA,NIM811,andother compounds can be explained by the presence of several cyclophilins compounds to cyclophilins was evaluated using a fluorescence L.A. Gaither et al. / Virology 397 (2010) 43–55 53 polarization displacement assay. The assay uses a CsA derivative pathways was performed by Gene Set Enrichment Analysis, GSEA which was covalently modified with the fluorophore Atto655 (ATTO- (Szustakowski et al., 2006). TECH GmbH) to generate the CsA-Atto655 probe. Briefly, a dose The complete data set is available at [email protected], series response curve (10 point, 3-fold dilution) was generated for GSE15935. compounds, cyclophilin and CsA-Atto655 probe were added subsequently and incubated for 30 min at room temperature. All assays siRNA-mediated down-regulation of mRNA were performed in 96-well plates at a final volume of 200 μl run in triplicate. Plates were read using a BMG PHERAstar (BMG Labtechnol- The siRNAs targeting cyclophilins (purchased from Dharmacon) or ogies) with a FP optic module (excitation: 620–40 nm, emission 1 and proteins identified in the chemogenomic approach (designed using 2: 750–50 nm). Milli-anisotropy values were converted to percent the Biopred algorithm (Huesken et al., 2005)) are listed in tables S1 inhibition, plotted vs. log compound concentration, and fitted with and4, respectively. Each siRNA was resuspended in siRNA buffer the 4-parameter logistic equation to determine the IC50.IC50 values (Dharmacon) to a stock concentration of 20 μM. The siRNAs were were converted to Ki values using a modified version of the Cheng– reverse transfected into Huh-luc/neo-ET cells in a 96-well dish using Prusoff equation under a competitive assumption. Lipofectamine 2000 (Invitrogen) in triplicate. Lipofectamine 2000 (0.25 μl/well) was diluted into 25 μl/well opti-MEM and incubated at Surface plasmon resonance assay room temperature for 5 min. The siRNA at 150 nM were diluted into opti-MEM (25 μl/well), and equal amounts of diluted lipofectamine The interaction between unmodified CsA or NIM811 with various 2000 and diluted siRNAs were mixed, incubated for 30 min at room cyclophilins was studied by immobilizing each individual cyclophilin temperature, and 50 μl was added to each well in a 96 well dish. Cells protein to a CM5 sensor chip using amine coupling chemistry. Data were plated at 8000 cells/100 μl DMEM (10 % FBS, 0.1 % NEAA) over obtained in a Biacore T-100 instrument (GE Healthcare) were the siRNA/lipofectamine 2000 mix in each well, giving a final siRNA analyzed using the BiaEvaluation software (GE Healthcare) to obtain concentration of 25 nM. Cells were incubated for 72 and 96 h and kon, koff, and KD values. assayed for replicon activity by luciferase using the BriteGlow reagent (Perkin Elmer), or HCV RNA copy number by quantitative real time Transcription profiling experiments PCR (as described in Ma et al., 2006) and for cell viability using the Cell Titer Glow kit (Promega). Naïve Huh7 cells and Clone A cells were plated at 106 cells per well in 6-well cell culture plates pre-incubated for 24 h at 37 °C (5% CO2) siRNA mediated decrease of target mRNA and then incubated in fresh medium or medium containing 5 μM CsA or NIM811 for 12, 24, and 48 h. The experiment was repeated twice to The effect of siRNAs on their target mRNA level was measured yield triplicate RNA samples for each condition. In a second using the Taqman Gene Expression Assay (Applied Biosystems) per experiment cells were treated with 1 and 5 μM CsA or NIM811 for manufacturer's instructions. Briefly, siRNAs were transfected in Huh7 48 h to analyze the effects of compound concentration. Total HCV subgenomic replicon cells in 96 well format as described above. cytoplasmic RNA was isolated from each sample using the Purescript Total RNA was isolated using the RNeasy96 Kit (Qiagen). cDNA RNA Purification System Cell and Tissue Kit (Gentra) and RNeasy® synthesis was carried out using the High Capacity cDNA reverse Mini Kit (Qiagen) according to the manufacturer's protocols. Integrity transcription kit (Applied Biosystems). Gene expression analysis was and purity of RNA was confirmed with the RNA 6000 Nano LabChip performed using Taqman probes and primers from Applied Biosys- system on Bioanalyzer 2100 (Agilent Technologies; Palo Alto, US). The tems in 384 well format. Each well contained 5 μl cDNA, 1 μl TaqMan double-stranded cDNA synthesis was carried out with 2 to 5 μg of total gene of interest probe, 1 μl β-actin probe and 10 μl Taqman gene RNA (SuperScript double-stranded cDNA synthesis kit; Invitrogen) expression Master Mix (Applied Biosystems). The reaction was run on according to the manufacturer's instructions. After purification an Applied Biosystems 7900HT Real Time PCR system (Applied (QIAquick, Qiagen AG), the cDNA was transcribed in vitro in the Biosystems). presence of biotinylated ribonucleotides (Bioarray High yield T7 DNA transcription kit, ENZO Life Sciences Inc.). The labeled cRNA was siRNA screen purified on an affinity resin (RNeasy, Qiagen AG), quantified and fragmented into strands of approximately 50–200 nucleotides in The siARRAY Protein Kinase siRNA Library (Dharmacon Technol- length. Hybridization to Affimetrix U133 Plus 2 chips was carried out ogies, catalog no. H-003500 of 779 smart pools) and Qiagen 4 siRNAs at 45 °C for approximately 18 h. The arrays were stained on GeneChip targeting 5000 druggable proteins (Human Druggable Genome siRNA Fluidics Workstation 450 and scanned on GeneArray Scanner 3000 Set X 1.0) were transfected into Huh-luc/neo-ET cells as previously according to manufacturer's technical manual (Affymetrix Inc.). The described (Rines et al., 2008; Hopkins and Groom, 2002). The total scanned image was converted into numerical values of the signal number of siRNA screened was 20,779 of which 20,000 were four intensity (Signal) and into categorical expression level measurement individual Qiagen synthesized siRNAs targeting 5000 druggable genes (Absolute Call) using the Affymetrix MAS 5.0 software. (Affymetrix and 779 Dharmacon smart pool kinases. The siRNAs were diluted and Inc.). The software scaled the average intensity of each chip to a target stamped out into 384 well tissue culture plates at a concentration of intensity of 150. The MAS 5.0 statistical algorithm parameters used to 206 nM diluted in 4 μl Opti-Mem (Invitrogen). Using 0.07 μlof attribute the Absolute Calls were set to 0.05 for Alpha 1, 0.065 for oligofectamine transfection reagent (Invitrogen) dilutions were Alpha 2 and 0.015 for Tau. Affymetrix selected QC parameters prepared in 4 μl Opti-Mem using the Flex Drop Multi-Dispenser “background,”“scaling factor,” 3′/5′ ratios of reference genes (e.g. (Perkin Elmer). The plates were incubated at room temperature for GAPDH, beta-actin), were checked for quality control purpose as to 20–30 min to allow complexes to form. After the incubation, 1500 detect arrays that need to be repeated. cells in 25 μl assay media were added per well using the Multidrop384 The processed arrays were quality checked using BioConductor (Thermo Labsystems). Cells were assayed for luciferase activity 72 h packages wrapped in a Perl script. The quality check included the post-transfection using the Bright Glo luciferase assay (Promega) and inspection of the intensity images, the MA plots for each array, the the siRNA collections were run in sets of n=2. The screening data was distribution of raw log2 expression values detected by PM probes and Z-score transformed to pick siRNAs that scored ≤50% below the the distribution of rma normalized log2 expression values in each median (≤0.5 or Z-score of −2) or ≥100% (≥2.0 or Z-score of 2) array relative to the all arrays in the experiment. The detection of above the median. Each well was subtracted from the median and 54 L.A. Gaither et al. / Virology 397 (2010) 43–55 divided by the standard deviation. The data reported in the tables are uk/IPI/) supplemented with sequences of HCV proteins as well as the absolute value of this number called 1D for one-dimensional common non-human contaminant proteins using Mascot (Matrix normalization. A value of 0.5 equals two standard deviations below Science). Validation of protein identifications and iTRAQ reporter ion- the median and 2 equals two standard deviations above the median. based quantification was performed using in-house developed For validation experiments each siRNA was resuspended in siRNA software. Only peptides unique to a set of peptides identifying a buffer (Dharmacon catalog no. B-002000-UB-015) to a stock concen- protein were used for relative protein quantification. iTRAQ reporter tration of 20 μM. 2.5 μl of each stock solution was diluted in 197.5 μlof ion intensities from all spectra matched to a given peptide species in Dharmacon Cell Culture Reagent (DCCR) (Dharmacon, catalog no. B- the total experiment were summed and protein fold changes were 004500-100) in a 96 well PCR plate (ABgene) to make a working 10 × determined from peptide values using a 2-sided linear regression stamp of 250 μM. Then 0.20 μl of Dharmafect1 (Dharmacon) was analysis. Minimum requirements for acceptance of quantified pro- diluted in 10 μl and added to each well of a 96 well tissue culture plate teins were at least four quantifiable spectra matching to unique (Costar). A 10 μl aliquot of each well in the 10× s tamp was added to peptides. the 96 well tissue culture plates containing lipid reagent and incubated for 20 min at room temperature to allow the complexes Acknowledgments to form. After the incubation, 6000 replicon cells in 80 μl of complete media was plated on top of the siRNA:lipid reagent complexes using We would like to thank Nicole Hartmann and her team for running the Multidrop384 (Thermo Labsystems). In parallel, the same the microchips for the RNA quantification, James Fang and Kwangho experiment was set up using 6000 replicon cells, to analyze if the Lee for modifying compounds to be used in the cyclophilin binding siRNAs also inhibit luciferase activity in non-replicon dependent cells assay and the chemoproteomic experiment. Many thanks to Alan (data not shown). Cells were incubated for 72 h at 37 °C and luciferase Abrams who created the view of the HCV life cycle in relation to activity was measured using the Bright-Glo luciferase assay (Pro- cyclophilins in human cells. mega) and cell viability was measured using CellTiter-Glo viability assay (Promega). References

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