inhibition of the interaction between ns3 protease and hcv ires with a small peptide: a novel...

original article© The American Society of Gene & Cell Therapy

Molecular Therapy vol. 21 no. 1, 57–67 jan. 2013 57

Recently, we have demonstrated that the protease domain of NS3 alone can bind specifically to hepatitis C virus (HCV) internal ribosome entry site (IRES) near the initiator AUG, dislodges human La protein and inhib-its translation in favor of viral RNA replication. Here, by using a computational approach, the contact points of the protease on the HCV IRES were putatively mapped. A 30-mer NS3 peptide was designed from the predicted RNA-binding region that retained RNA-binding ability and also inhibited IRES-mediated translation. This pep-tide was truncated to 15 mer and this also demonstrated ability to inhibit HCV RNA-directed translation as well as replication. More importantly, its activity was tested in an in vivo mouse model by encapsulating the peptide in Sendai virus virosomes followed by intravenous delivery. The study demonstrates for the first time that the HCV NS3-IRES RNA interaction can be selectively inhibited using a small peptide and reports a strategy to deliver the peptide into the liver.

Received 28 November 2011; accepted 5 July 2012; advance online publication 21 August 2012. doi:10.1038/mt.2012.151

IntroductIonHepatitis C virus (HCV) is a single-stranded positive sense RNA virus belonging to Flaviviridae family.1 The 9.6-kb genome con-sists of a 5′ untranslated region (5′UTR), a single open reading frame encoding the viral proteins followed by a 3′ untranslated region (3′UTR).2 It is the primary causative agent of non-A, non-B hepatitis, which often leads to liver cirrhosis and hepatocellular carcinoma.3,4 HCV RNA translation occurs by a cap-independent mechanism. The internal ribosome entry site (IRES) located in the 5′UTR allows ribosomes to assemble directly on a site located a few nucleotides upstream of the initiator AUG and initiate poly-protein synthesis.5–9 Several cellular factors interact with HCV RNA and facilitate IRES-mediated translation. Nonstructural protein 3 (NS3), a multifunctional enzyme, is necessary for poly-protein processing and also has helicase activity.

Recently, we have shown that dislodging the human La pro-tein, a host factor, from the GCAC motif near the initiator AUG of the HCV IRES by the protease domain of the NS3 protein, leads to translation inhibition in favor of RNA replication.10 These pro-teins have been shown to share a binding region in stem loop IV (SLIV) of the IRES near the initiator AUG. It is possible that, after dislodging La, the ribosomal complex dissociates and the repli-cation machinery can move freely along the RNA allowing RNA replication to occur. Since, NS3 has a role in translation as well as replication; it is a potent antiviral target.

HCV infection is a major health concern. Since the discovery of the virus, considerable progress has been made in the under-standing of the virus life cycle and in development of therapies. Introduction of treatment with pegylated interferon and ribavirin increased the proportion of patients achieving sustained antivi-ral response. Despite this, the toll of patients suffering from HCV infection continues to rise and a proportion fails to respond to available therapy.11–13 The response of patients also depends on the viral genotype and non responders to previous interferon-based therapies often fail after retreatment.14 Moreover, side-effects of current therapies add to the complexity.

Theoretically, each step in the viral life cycle is suitable for anti-viral intervention and various viral proteins have been considered as antiviral targets. This includes the NS3 protein, and the NS3 protease inhibitors, telaprevir and boceprevir, which were recently licensed by the FDA. To date, most NS3/4A inhibitors in clinical trials are peptide-based and represent cleavage products targeting the protease cleavage site. In addition, valopicitabine was devel-oped against the HCV RNA-dependent RNA polymerase15 and there are reports of attempts to inhibit HCV replication by sev-eral different agents including peptides, small RNA decoys, DNA or RNA aptamers, small interfering RNAs, short hairpin RNAs, ribozymes, DNAzymes etc.16–20 In spite of these efforts, the virus can often develop resistance to these compounds due to genetic heterogeneity resulting in selection of genomes already having a resistant imprint.21 Therefore, it is essential to identify newer drug targets and develop more efficacious less toxic anti-HCV compounds.

The first two authors contributed equally and should be considered as joint first authors.Correspondence: Saumitra Das, Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore-560012, India, E-mail: [email protected]

Inhibition of the Interaction Between NS3 Protease and HCV IRES With a Small Peptide: A Novel Therapeutic StrategyUpasana Ray1, Chaitrali L Roy1, Anuj Kumar1, Prashant Mani2, Agnel P Joseph3, G Sudha3, Debi P Sarkar2, N Srinivasan3 and Saumitra Das1

1Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India; 2Department of Biochemistry, University of Delhi, South Campus, New Delhi, India; 3Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India

http://www.nature.com/doifinder/doi:10.1038/mt.2012.151

mailto:[email protected]

58 www.moleculartherapy.org vol. 21 no. 1 jan. 2013

© The American Society of Gene & Cell Therapy

resultsthe rnA-binding region of ns3 protease is located in the c-terminal halfPreviously, it has been demonstrated that the NS3 protease binds to the HCV IRES in the SLIV region.10 A prediction of probable RNA-binding sites was carried out using RNABindR, BindN, and Pprint servers (Figure 1a). Based on these predictions and the elec-trostatic surface potential calculation studies, the NS3 protease was modeled with the HCV SLIV region of the IRES (Figure 1b and Supplementary Figure S1a,b). It appeared from the predictions that the RNA-binding residues were concentrated in the C-terminal half. Moreover, multiple sequence alignment of the NS3 protease domain from various HCV genotypes showed conservation of the predicted RNA-binding residues (Supplementary Figure S2). NS3pro was consequently expressed as two regions, ΔN-NS3pro and ΔC-NS3pro (Figure 2a). These proteins were then used in gel retarda-tion assay with α[32P]-labeled HCV IRES RNA probe (Figure 2b and Supplementary Figure S3a). Increasing concentrations of ΔN-NS3pro showed a dose-dependent shift indicating that the C-terminal region

interacted with the HCV IRES RNA whereas, the binding was insig-nificant with ΔC-NS3pro. We also showed that NS3 protease inhibited IRES-mediated translation upon RNA binding.10 Hence, the effect of ΔN-NS3pro and ΔC-NS3pro on IRES-mediated translation was deter-mined in vitro using a bicistronic construct in which firefly luciferase translation is controlled by the HCV IRES and renilla luciferase is controlled by cap-dependent translation (Figure 2c). An in vitro translation assay showed that ΔN-NS3pro, by virtue of its binding to the HCV IRES, also inhibited IRES-mediated translation. In sum-mary, the results showed that the RNA-binding residues of the NS3 protease are predominantly located in the C-terminal half and this region is capable of inhibiting HCV IRES-mediated translation.

A 30-mer peptide derived from Δn-ns3pro binds HcV Ires and inhibits HcV Ires-mediated translationWhile the 3-D structure of the NS3 protein in unbound form is available (PDB code: 1CU1), the structure of NS3 bound to HCV IRES has not been determined yet. Therefore, a 3-D model of the protease domain complexed with SLIV of HCV IRES was

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Figure 1 Prediction of probable rnA-binding residues of ns3 protease. (a) Prediction of probable RNA-binding residues of NS3 protease was car-ried out by submitting the NS3 protease sequence to RNABindR, BindN, and Pprint servers. Plus signs indicate the predicted RNA-binding residues. The shaded area indicates the region where the predicted RNA-binding residues were more concentrated. The 30-mer peptide (NS3proC-30) was derived from this region. (b) The NS3 protease was modeled along with stem loop IV region of hepatitis C virus (HCV) internal ribosome entry site (IRES).

Molecular Therapy vol. 21 no. 1 jan. 2013 59


generated. Arg 123, Lys 136, Ala 157, Ala 162, Asp 168 were found to interact with SLIV by hydrogen bonding. Many other hydro-gen-bondable groups, such as those from side chains of Ser 133 and Tyr 134, are poised for interaction with RNA. Further, apolar side chains such as Ile 132 and Leu 135 could form favorable Van der Waals interaction with the HCV RNA. The top scoring mod-eled complex was generated by PatchDock which considers the geometric score suggesting good shape complementarity, inter-face area size, atomic contact energy and potential binding site information along with various favorable interactions. This model suggested reasonable goodness of fit rendering the NS3 protease–SLIV complex stable.

However, NS3 exists as a bifunctional enzyme with a heli-case domain in addition to the protease domain (PDB id: 1CU1). Using structural superposition by ProFit, the model of the NS3 protease domain bound to RNA was extrapolated to model the full-length NS3 (PDB code: 1CU1)–RNA complex. This model also revealed that the RNA can be accommodated in the presence of helicase domain between the interface of the protease and the

helicase domains. This is facilitated by the long and flexible linker connecting these two domains.

Based on the predictions and modeling (Figure 1 and Supplementary Figure S1a,b), a stretch of 30 amino acid resi-dues of NS3 (114–143) was predicted to interact with SLIV. Consequently, a 30-mer peptide (NS3proC-30) was designed from the C-terminal half of the NS3 protease (Figure 2d,e and Supplementary Figure S1c–e). This peptide showed significant binding to HCV IRES in a UV crosslinking assay (Figure 3a and Supplementary Figure S3b) but a mutant NS3proC-30 pep-tide in which Ser 138 and Ser 139 were replaced with alanines showed reduced binding activity (Figure 3a). The affinity of the NS3proC-30 peptide was also determined using a filter-binding assay and was determined to be ~22 μmol/l (Figure 3b). The addition of the wild-type peptide to an in vitro translation assay significantly inhibited translation in a concentration-dependent manner (Figure 3c). According to the predictions, the C-terminal half of this 30-mer peptide binds more proximal to the SLIV of the HCV IRES than the N-terminal half which showed fewer amino

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Figure 2 c-terminus of the ns3 protease interacts with the hepatitis c virus (HcV) internal ribosome entry site (Ires) and inhibits HcV Ires-mediated translation. (a) Schematic showing the generation of NS3 protease fragments. (b) The N-terminal half (ΔC-NS3pro) (lanes 2–6) and C terminal half (ΔN-NS3pro) (lanes 7–11) of NS3pro were bound to [α32P] UTP-labeled HCV IRES RNA and the mixture was analyzed by gel retardation assay. Lane number 1 indicates the no protein control and the arrow indicates the position of the shifted band. (c) In vitro transcribed capped bicis-tronic RNA (shown as a schematic above the panel) was translated in vitro in the absence or presence of increasing concentrations of either NS3pro, ΔN-NS3pro, or ΔC-NS3pro. The results were analyzed by luciferase assay. Percent luciferase activities corresponding to Rluc (white bar) and Fluc (gray bar) values were plotted against the protein concentrations. (d) Schematic representation of the three NS3pro derived peptides. (e) Computational model of 30-mer peptide bound to stem loop IV (SLIV) region of HCV RNA.



acid residues that interacted with SLIV. Although the N-terminal half is able to bind HCV RNA, the purpose is probably to serve as a stabilization factor. Consequently, the 30-mer peptide was then synthesized in two halves, the NS3proC-N15 and the NS3proC-C15 (Figure 2d and Supplementary Figure S1e).

Both peptides showed considerable binding to HCV IRES in an UV crosslinking assay (Figure 3a and Supplementary Figure S3b), however, in a filter-binding assay the NS3proC-C15 peptide showed higher affinity (~29 μmol/l) (Figure 3b and Supplementary Figure S4a). The scrambled NS3proC-C15 peptide

did not show appreciable binding in these assays (Figure 3b and Supplementary Figure S3b). The NS3proC-N15 peptide structure was found to be rigid and contain a β hairpin turn which might reduce interactions with RNA. The NS3proC-C15 peptide on the other hand has an alpha helical structure formed by the region 131–137 (PISYLKG) that might contribute to most of the RNA-binding activity.

In the in vitro translation assay the NS3proC-C15 peptide showed significant inhibition of IRES-mediated translation whereas the scrambled NS3proC-C15 peptide did not inhibit

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Figure 3 Peptides derived from ns3 protease bind the hepatitis c virus (HcV) internal ribosome entry site (Ires) and inhibit HcV Ires-mediated translation. (a) [α32P] UTP-labeled HCV IRES RNA was UV-crosslinked to increasing concentrations of 30-mer (lanes 2–3), mutant 30-mer (lanes 4–5), N15 (lanes 6–7), and C15 (lanes 8–9) as indicated. The reactions were analyzed on a Tris-tricine 15% gel. (b) Filter-binding assay. [α32P] UTP-labeled HCV IRES RNA was incubated with increasing concentrations of 30-mer, N15, C15, and scrambled C15 peptide. The RNA–protein complex was captured by binding to nitrocellulose filters. The percentage of RNA retained was plotted against the peptide concentrations used. (c) In vitro transcribed capped bicistronic RNA was in vitro translated in the absence or presence of increasing concentrations of either 30mer, N15, or C15 peptides. Luciferase assay was performed. Percent luciferase activities corresponding to Rluc and Fluc values were plotted against the peptide concentrations. White bar indicates Rluc and gray bar indicates Fluc activity. (d) In vitro transcribed capped bicistronic RNA was translated in rabbit reticulocyte lysate in the absence or presence of increasing concentrations of C15 peptide. IC50 was attained at a concentration of around 5 µmol/l whereas IC90 was obtained in between 60 and 80 µmol/l concentration. (e) In vitro transcribed capped bicistronic RNA was in vitro translated in the absence or presence of increasing concentrations (5, 10, 20, 40, 60, 80 µmol/l) of scrambled NS3proC-C15 peptide. Luciferase assay was performed. Percent luciferase activities corresponding to Rluc (White bar) and Fluc (gray bar) values were plotted against the peptide concentrations.



translation (Figure 3c and e). The IC50 and IC90 values for the NS3proC-C15 peptide were determined (Figure 3d) and shown to be 5 μmol/l and 60–80 μmol/l, respectively. Furthermore, the peptide showed no inhibition of other viral IRES exemplified by EMCV IRES (Supplementary Figure S4b).

Validation of the modeled complex by a mutant ns3 peptide with reduced binding ability to slIV rnAThe structural contexts of critical residues of the NS3 peptide which are involved in the interaction with SLIV RNA were ana-lyzed using the modeled complex. The model revealed that Ser 138 of NS3proC-C15 stabilize the conformation of the backbone where a critical Lys 136 interacts with the RNA. According to the model, the side chain (OG) atom of Ser 138 is hydrogen bonded with the major chain carbonyl oxygen of Leu 135 (Supplementary Figure S5). This interaction is crucial for the proper positioning of Lys 136 which was shown to be a RNA-binding residue in a previous study. The absence of this interaction may increase the conforma-tional entropy of the region spanning residues 135-138, thereby compromising on the structural constraint on Lys 136 to orient itself appropriately to interact with RNA. The flexibility of this region could be further enhanced by the Gly 137 residue following Lys 136 that has higher conformational entropy due to the absence of a side chain. In order to explore the validity of this assertion, a mutant in which Ser 138 is replaced by Ala was designed. In this mutant the side chain-main chain interaction (which involves Ser 138 in the WT) in the vicinity of the RNA-interacting Lys 136 is expected to be absent.

the human la protein can rescue the ns3proc-c15 peptide-mediated inhibition of Ires-mediated translationWe showed previously that La protein binds near the initiator AUG and stimulates IRES-mediated translation.22 It was also shown that the NS3 protease and the La protein bind at similar places on SLIV.10 Since, the NS3proC-C15 peptide was derived from the RNA-binding region of the NS3 protease, the contact points of the peptide were then confirmed to be in similar location as evident from a toe-printing assay (Figure 4a,b). Addition of La protein in the in vitro translation reaction showed to reverse the translation inhibition due to NS3proC-C15 peptide (Figure 4c). However, a mutant La protein (P4La)16 that is inefficient in bind-ing to the HCV IRES failed to rescue the inhibition (Figure 4c). Moreover, a competitive UV crosslinking assay using radiolabeled HCV SLIV RNA showed that the NS3proC-30 and the NS3proC-C15 peptides could efficiently compete out La binding to SLIV RNA (Figure 4d).

ns3proc-c15 peptide inhibits HcV Ires-mediated translation in mammalian cellsTo examine the effect of the peptides on HCV IRES-mediated translation in mammalian cells the NS3proC-C15, NS3proC-N15, and scrambled NS3proC-C15 peptides were tagged with hexa argin-ine which is known to facilitate peptide transduction.16 Huh 7 cells were transfected with the HCV bicistronic construct followed by the addition of 10 μmol/l of each peptide and luciferase activities were measured 24 hours post-transduction. NS3proC-C15 showed

around 60% inhibition of IRES-mediated translation (Figure 5a). Increasing concentrations of NS3proC-C15 peptide resulted in 80% inhibition of translation (Figure 5b).

Furthermore, the effect of the peptides on HCV RNA replica-tion was confirmed by transient transfection of Huh7 cells with HCV monocistonic replicon RNA followed by peptide transduc-tion. Twenty four hours post-transduction, total RNA was iso-lated and the level of HCV negative strand was measured using real time RT-PCR. The NS3proC-C15 peptide showed greater inhibition of HCV RNA level compared to NS3proC-N15 pep-tide, and the scrambled NS3proC-C15 peptide showed no inhibi-tion (Figure 5c). Further, to investigate the changes in the level of HCV protein expression, western blot analysis was performed using anti-NS5B antibody. NS3proC-C15 peptide showed consid-erable decrease in NS5B protein level compared to NS3proC-N15 or the scrambled peptide (Figure 5d).

Similarly, the effect of the peptides on the HCV RNA repli-cation in the infectious cell culture system was confirmed by transient transfection of Huh7.5 cells with infectious JFH1 RNA followed by the peptide transduction. The NS3proC-C15 peptide showed considerable inhibition of HCV negative strand synthesis (Figure 5e) and also showed considerable decrease in the NS5B protein level in the western blot analysis compared to controls (Figure 5f).

To confirm that inhibition of replication of HCV RNA by the NS3proC-C15 peptide was not due to degradation of the RNA, the monocistronic replicon RNA was transfected in Huh7 cells fol-lowed by the addition of peptide. The 5′UTR and the 3′UTR were amplified by RT-PCR. No reduction was observed in the intensity of the amplicons ruling out the possibility of 3′end directed deg-radation of the RNA (Supplementary Figure S6a,b). In addition, there was no apparent decrease in the cellular protein profile as observed by in vivo labelling using 35S methionine in the presence and absence of the NS3proC-C15 peptide (Supplementary Figure S7a). Although these data suggested that the NS3proC-C15 peptide was not toxic, its toxicity was formally tested in MTT assay and showed no substantial reduction in cell viability after addition to Huh7 cells (Supplementary Figure S7b).

ns3proc-c15 peptide inhibits HcV Ires-mediated translation in miceAbove results showed that the NS3proC-C15 peptide bound to HCV SLIV and inhibited HCV IRES-directed translation. Consequently, the peptide also inhibited HCV RNA replication in cell culture system. To determine whether the peptide is able to inhibit HCV IRES-mediated translation in vivo, Sendai virus-based virosomes were used for preferential delivery of the peptides into the liver of mice. To determine whether the peptide encap-sulated in virosomes is able to enter liver cells efficiently, FITC-conjugated NS3proC-C15 peptide was encapsulated in F-virosomes derived from Sendai virus. The F-protein on the virosome inter-acts specifically with the asialoglycoprotein receptor on the sur-face of the mouse hepatocyte thereby leading to membrane fusion and release of the contents in the cytoplasm.

Before inoculation of the mice, encapsulation of the peptide into the virosome was confirmed by western blot analysis using anti-NS3 antibody (Supplementary Figure S8a). Furthermore,



fusion-mediated delivery of the virosome was confirmed by incu-bating Huh7 cells with the virosomes for 2 hours at 37 °C followed by fluorescence microscopy 24 hours later. The strong signal obtained as shown by green fluorescence (Supplementary Figure S8b–d) indicates that the virosomes could efficiently deliver the peptides.

Using the same experimental conditions, the HCV bicistronic con-struct and the peptides were encapsulated in F-virosomes and deliv-ered in Huh7 cells. Luciferase expression was measured to examine the effect of the peptide on HCV IRES-mediated translation. These results suggested ~60% reduction in translation (Figure 6a).

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Figure 4 ns3pro c-c15 peptide competes with human la protein for binding to hepatitis c virus (HcV) stem loop IV (slIV) rnA. (a) Toe-printing assay: HCV SLIV RNA was incubated with increasing amounts of the proteins/peptides as mentioned and analyzed by primer extension. Lane 1 shows no protein control. Lanes GATC shows the DNA sequencing ladder corresponding to HCV SLIV RNA obtained by using the same end-labeled primer. (b) Schematic of HCV SLIV indicating the toe-prints obtained. (c) Rescue of translation inhibition by La protein. In vitro transcribed capped bicistronic RNA was in vitro translated in the absence or presence of NS3proC-C15 peptide along with either wild-type La or mutant La (P4La) protein. Luciferase assay was performed. Percent luciferase activities corresponding to Rluc (white bar) and Fluc (gray bar) values were plotted against the protein concentrations. (d) Competition UV crosslinking assay. [α32P] UTP-labeled HCV SLIV RNA was UV-crosslinked with La protein in the presence of 100- or 200-fold molar excess of NS3proC-30 (lanes 3 and 4), NS3proC-N15 (lanes 5 and 6), NS3proC-C15 (lanes 7 and 8), and NS3pro (lanes 9 and 10). Lane 1 represents no protein control and lane 2 shows binding due to La protein alone.



Figure 5 effect of hexa arginine-tagged ns3 peptides on hepatitis c virus (HcV) rnA translation and replication ex vivo. (a) Huh7 monolayer cells were transfected with 2 µg of HCV bicistronic DNA. Four hours later, 10 µmol/l of either Arg-NS3proC-N15, Arg-NS3proC-C15, or Arg-NS3proC-Sc15 peptides were added to the cells. The cells were harvested after 24 hours, lysed and luciferase activities were measured using the dual luciferase assay system. The Rluc (white bar) and Fluc (gray bar) activities were plotted as fold decrease in presence of the peptide with respect to the corresponding control (in the absence of peptide) plotted as 100. (b) Using similar experimental conditions as described in panel a, Arg-NS3proC-C15 was added in increasing concen-trations (5, 10, 20, 40, 60 µmol/l) followed by luciferase assay. (c) Schematic representation of HCV monocistronic replicon (adapted from ref. 33). Huh7 cells were transfected with HCV monocistronic replicon RNA (adapted from ref. 33). Four hours later Arg-NS3proC-N15 (10 µmol/l), Arg-NS3proC-Sc-C15 (10 µmol/l), and Arg-NS3proC-C15 (5, 10, 20 µmol/l) peptides were transduced. Twenty four hours post-transduction, total cellular RNA was isolated and real time reverse transcription (RT)-PCR was performed to detect the HCV negative strand RNA level. GAPDH was used as an internal control. (d) For the experiment performed in panel c, cells were lysed and western blot was done using anti-NS5B antibody. β-Actin was used as an internal control. (e) Schematic representation of HCV pJFH1 construct (adapted from ref. 34). Huh7.5 cells were transfected with infectious JFH1 RNA. Four hours later, Arg-NS3proC-N15 (10 µmol/l), Arg-NS3proC-ScC15 (10 µmol/l), and Arg-NS3proC-C15 (10, 20 µmol/l) peptides were transduced. Twenty four hours post-transduction, total cellular RNA was isolated and real time RT-PCR was performed to detect the HCV negative strand RNA level. GAPDH was used as an internal control. (f) For the experiment performed in panel e, cells were lysed and western blot was done using anti-NS5B antibody. β-Actin was used as an internal control.

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To examine the in vivo efficacy of peptide inhibition, F-virosomes loaded with the HCV bicistronic construct the peptide/s was injected into BALB/c mice via the tail vein (Supplementary Figure S9). Two days postinjection, the animals were sacrificed, the hepatocytes were isolated and luciferase activity was measured (Supplementary Figure S10). The NS3proC-C15 peptide showed ~52% inhibition of HCV IRES-mediated translation, but the scrambled peptide showed no inhibition (Figure 6b). The NS3proC-C15 peptide showed no inhi-bition of other viral IRES exemplified by EMCV IRES (Figure 6c) thus confirming the in vivo specificity of the peptide. It appears that the NS3proC-C15 peptide when delivered to hepatocytes in which protein expression is controlled by the HCV IRES, is able to inhibit IRES-directed translation and thus reduce the level of expression of the reporter molecule.

dIscussIonThe HCV RNA-dependent RNA polymerase (NS5B) and NS3 pro-tease are the most common targets for antiviral agents. A major reason is the considerable sequence conservation of these pro-teins across the genotypes. Nevertheless, the emergence of escape

variants has been reported. The sequence of the SLIV adjacent to the iAUG (320–359) is highly conserved. Thus, if the interaction between the NS3 protein and the highly conserved viral RNA is targeted, the frequency of viral escape mutants is predicted to be very low. Moreover, the NS3 peptide bound to the HCV IRES would interfere with the translation/replication switch and act as a dominant negative inhibitor of viral RNA translation. In fact, the NS3proC-C15 peptide derived from the RNA-binding region of the NS3 protease did show significant inhibition of translation and replication of HCV RNA. However, at this point it is not clear if the inhibition of replication is the consequence of translation inhibition. RNA toe printing revealed that the NS3proC-30 and the NS3proC-C15 peptides bound near the iAUG.

An interaction at this region of the HCV SLIV will inhibit ribosomal movement on the RNA and also prevent La protein binding at the GCAC region. Thus, the peptides could inhibit HCV IRES translation in two ways, firstly by dislodging La pro-tein and secondly by blocking 40S-ribosome interaction near the initiator AUG. The concentration of peptide required to dislodge the La protein was higher than that of full-length NS3 protease. It

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C NS3proC-C15 NS3proC-ScC15 C NS3proC-C15 NS3proC-ScC15

Figure 6 Virosome mediated delivery of peptide: the effect on hepatitis c virus (HcV) translation. (a) Huh7 cells were incubated with loaded F-virosomes containing 4 µg of HCV bicistronic construct in the presence or absence of the NS3proC-C15 peptide or scrambled NS3proC-C15 peptide in serum-free medium at 37 °C. Two hours later, 10% serum-containing Dulbecco’s modified Eagle’s medium (DMEM) was added and cells were further incubated for 24 hours and the results analyzed by the luciferase assay. (b) HCV bicistronic construct with NS3proC-C15 or scrambled NS3proC-C15 peptide was loaded into F-virosomes and injected into the tail vein of male BALB/c mice. Two days postinjection, animals were sacrificed, hepatocytes were isolated and luciferase activity was performed. (c) The EMCV bicistronic construct and the NS3proC-C15 peptide or scrambled NS3proC-C15 peptide was loaded inside F-virosomes. Male BALB/c mice were injected into the tail vein with these loaded virosomes. Two days postinjection, the animals were sacrificed, the hepatocytes were isolated and luciferase activity was measured.



is possible that the peptide might be capable of dislodging La pro-tein partially but the fact that it can bind near the initiator AUG might be an additional contributing factor to make the translation inhibition more effective.

Furthermore, we analyzed the interacting residues using in silico approach. The prediction showed a distribution of RNA-binding residues throughout the protease domain with a preferen-tial aggregation in the C terminal half. Interestingly, the C terminal half showed considerable RNA binding and translation inhibition. Thus the NS3proC-30 peptide derived from the C-terminal RNA-binding region of the NS3 protease effectively mimic the protease binding to the IRES element. In fact, structural analysis of this peptide indicated the presence of extensive hydrogen bonds that might make this 30mer peptide highly stable even in isolation. More importantly, a shorter peptide comprising of the C terminal part of the NS3proC-30 peptide retained the RNA-binding ability and showed significant inhibitory activity. Based on the structure function information, it will be interesting to design a peptidomi-metic for these peptides to increase stability and bioavailability.

To test the efficacy of the peptides ex vivo, the 11 mer trans-duction domain of the HIV Tat protein was utilized that is well known to mediate protein transductions. This domain contains six arginine residues which, when tagged upstream of a short peptide, can efficiently mediate transduction.16 Arginine-tagged NS3proC-C15 peptide exhibited considerable inhibition of HCV RNA translation and consequent inhibition of HCV RNA repli-cation compared to the NS3proC-N15 peptide. The inhibition of HCV RNA replication by the NS3proC-C15 peptide is further cor-roborated in the inhibition of HCV protein synthesis. It is possible that the NS3proC-N15 peptide might not maintain its structure ex vivo and could not efficiently inhibit HCV RNA function in an intracellular location.

The NS3proC-C15 peptide was tested in a mouse model by pref-erential delivery to the liver using Sendai virus-based F-virosomes simultaneously with a reporter construct. These F-virosomes are reconstituted envelopes of Sendai virus containing only the fusion glycoprotein (F-protein). F-virosomes have been shown to undergo liver-specific membrane-fusion through its terminal galactose moiety by interacting with the asialoglycoprotein recep-tors present on the surface of the hepatocytes.23–25 Our results showed more than 50% inhibition of HCV IRES translation at 10 μmol/l concentration. To increase the effectiveness of the pep-tide it will be essential to work on its stability.

In our earlier study, we targeted the human La protein, an important host factor in the HCV life cycle. This protein binds to the HCV SLIV region and enhances IRES-mediated translation.26,27 A small peptide (LaR2C-N7) derived from the RNA-binding region of La protein was shown to inhibit HCV RNA-directed transla-tion and replication of HCV RNA.16 Interestingly, the LaR2C pep-tide was also shown to bind predominantly to the GCAC motif. Since both the NS3 peptide and LaR2C bind to a similar region and prevent binding of either the host factor or the viral protein, it would be interesting to investigate the effect of these inhibitors in combination.

As the genetic variability of the virus enables it to develop anti-viral resistance, it becomes a challenge to develop specific HCV inhibitors. Consequently, combination therapy is likely to be more

effective. We believe that a combination of these peptides might inhibit the viral RNA functions in a more aggressive manner to develop an effective antiviral therapeutic strategy.

MAterIAls And MetHodsComputational model of protease bound to HCV 5′UTR RNA. RNA-binding residues in NS3 protease were predicted by RNABindR,28 BindN,29 and Pprint.30 The predictions of RNA-binding residues were based on the amino acid environment, biochemical features and the pro-pensity of various amino acid residues for RNA-binding residues that are calculated from the known 3-D structures of protein–RNA or protein–DNA complexes. Residues that were predicted to bind to RNA by at least two of these methods and the side chains that were accessible at the sur-face were mapped onto the protease structure. These residues were also checked for conservation among various subtypes of HCV using multiple sequence alignment by clustalW.

RNA binding of the NS3 protease domain was studied by analyzing the surface electrostatic potential using Delphi31 and the solvent accessibility of the residues in a positively charged patch of NS3 protease that might be involved in RNA binding. The SLIV model in HCV RNA was generated using the MC FOLD/MC SYM pipeline.32 Patchdock was used to dock the HCV RNA with the NS3 protease based on shape complementarity. DISPLAR and PPI-PRED servers were also used to predict the nucleic acid (DNA) binding property in the NS3 protease, based on information such as secondary structure, hydrogen bonding potential and solvent accessibility. The PBALIGN tool implemented in the PBE server was used to find protein structures having local structural similarities with the NS3 protease. RNA and DNA binding proteins were manually chosen from the top hits (Z-score >2.5). The structures of each of these hits were analyzed carefully to identify the nature and geometry of the protein-nucleic acid interaction.

Alanine scanning mutagenesis experiments carried out previously along with foot-prints and toe-prints were considered while docking the SLIV at the catalytic site of NS3 protease which showed preference for binding of double helical RNA. PyMol and Chimera were used to visualize the 3-D structures of the protein and RNA in this study.

In vitro transcription and translation. mRNAs were transcribed from different plasmid constructs by run-off transcription reactions using T7 RNA polymerase (Promega). Radiolabeled mRNAs were generated in similar reactions supplemented with α [32P]UTP.

In vitro translation of capped bicistronic RNA was carried out in a reaction containing nuclease treated rabbit reticulocyte lysate (RRL) (Promega), amino acid mix and RNase inhibitor (as per manufacturer’s protocol). Renilla and firefly luciferase activities were measured using the Promega dual luciferase assay system.

UV crosslinking assay. The α[32P]UTP-labeled RNA of interest was incu-bated with protein/peptide at 30 °C for 15 minutes in RNA-binding buf-fer (containing 5 mmol/l HEPES pH 7.6, 25 mmol/l KCl, 2 mmol/l MgCl2, 3.8% glycerol, 2 mmol/l DTT, and 0.1 mmol/l EDTA). The reaction mix-tures were then UV-crosslinked using short wavelength UV light for 15 minutes. The reaction mixtures were then treated with RNase A (Sigma). The protein nucleotide complexes were thereafter denatured in 1X SDS dye and resolved in SDS-PAGE followed by phosphor imaging.

Filter-binding assay. The α[32P]UTP-labeled HCV RNA was incubated with the protein/peptide of interest at 30 °C for 15 minutes in RNA-binding buffer. The reactions were loaded on nitrocellulose filters previously equili-brated with RNA-binding buffer, the filters were washed thrice with RNA-binding buffer, dried and retained counts were measured in a scintillation counter. The protein concentration was plotted against the percentage of RNA retained. Each experiment was repeated in triplicate and the mean value along with standard error was measured.



Gel retardation assay. α[32P]UTP-labeled HCV SLIV RNA was incubated with the protein of interest at 30 °C for 20 minutes in RNA-binding buf-fer. The reactions were loaded on 4% polyacrylamide gel and separated by electrophoresis at 12 mA in 4 °C until the dye front reached the lower half of the gel. The gel was then fixed and dried, and the results analyzed by phosphorimaging.

Transient transfection and peptide transduction. Huh7 and Huh7.5 monolayer cells (80% confluent) were transfected with HCV monocis-tronic replicon 2a33 and JFH1 RNA,34 respectively using Lipofectamine 2000 reagent (Invitrogen) in antibiotic free medium. Four hours post-transfec-tion, 6× Arginine-tagged peptide was added in Dulbecco’s modified Eagle’s medium and supplemented with 10% fetal bovine serum. The cells were harvested after 24 hours using TRI reagent (Sigma) for total RNA isolation and RT-PCR. To measure IRES-mediated translation, the cell lysates were prepared using passive lysis buffer (Promega) and luciferase activity was measured using the Luciferase assay reagent (Promega).

Toe-printing assay. Unlabeled HCV SLIV RNA was incubated in the pres-ence or absence of increasing concentrations of protein/peptide at 30 °C for 20 minutes and then annealed to an end-labeled primer (75 fmoles) with sequence complementary to the 3′ end of the RNA. The RNA–protein complex was reverse-transcribed with 3 units of AMV-RT (Promega) at 45 °C for 30 minutes followed by denaturation of AMV-RT at 95 °C for 3 minutes. The cDNAs were then subjected to phenol chloroform extrac-tion and ethanol precipitation. The dried pellets were dissolved in nuclease free water and resolved on 7 mol/l urea–8% acrylamide gel. In parallel, a sequencing reaction using the same end-labeled primer was examined by electrophoresis to indicate the exact position of the RT stops.

Real-time RT-PCR. Total RNA was isolated using TRI reagent (Sigma) and reverse transcribed by MMLV RT (Fermentas) using HCV 5′ primer and GAPDH 3′ primer to detect negative strand of HCV RNA. cDNA was used for PCR using a real time assay mixture (Finnzymes) as per the manufac-turer’s instructions, and the data was analyzed by using ABI-Prism’s real-time PCR machine. GAPDH was used as an internal control.

Western blotting. Protein concentrations of cell lysates were assayed by Bradford assay (Bio-Rad) and equal amounts of cell extracts were sepa-rated by 10% SDS-PAGE and transferred to nitrocellulose membrane (PALL Life Sciences). Samples were then analyzed by western blotting using rabbit polyclonal anti-NS5B antibody (ab35586; Abcam, Cambridge, MA) followed by secondary antibody (horseradish peroxidise conjugated anti-rabbit IgG; Sigma). The blots were developed using ECL techniques (Amersham Pharmacia Biotech). Rabbit monoclonal anti-β-actin per-oxidase antibody (Sigma) was used as control for equal loading of cell extracts.

Fusion-mediated delivery of plasmids and peptides to cells in culture. Monolayers of Huh7 cells were incubated with loaded F-virosomes (0.3 mg F-protein) containing 4 μg of the bicistronic HCV plasmid in the presence or absence of NS3proC-C15 or scrambled NS3proC-C15 peptide in serum-free medium. Two hours post-fusion, 10% serum-containing Dulbecco’s modified Eagle’s medium was added and cells were then incubated for 24 hours before analysis of luciferase expression.

Administration of loaded F-virosomes to mice. HCV or EMCV bicistronic construct and NS3proC-C15 peptide or scrambled NS3proC-C15 was loaded inside F-virosomes. Male BALB/c mice weighing ~36 g were injected (two animals per group) into the tail vein with these loaded virosomes. Two days postinjection, the animals were sacrificed, hepatocytes were isolated and luciferase activity was measured.

Animal experiment. The mice experiments were carried out as per criteria outlined in the guidelines approved by Government of India.

suPPleMentArY MAterIAlFigure S1. (a) Electrostatic surface potential calculation for the pro-tease domain (using Delphi) showing a positively charged patch.Figure S2. Comparison of NS3 protease amongst different geno-types: Multiple sequence alignment of the NS3 protease domain from various genotypes of HCV shows the conservation of the predicted RNA-binding residues which are highlighted in yellow.Figure S3. (a) Electrophoretic mobility shift assay: The full-length NS3 protease (NS3pro) and N terminal half (ΔC-NS3pro) (lane2-6), C ter-minal half (ΔN-NS3pro) of NS3pro were bound to [α32P] UTP-labeled HCV IRES RNA and the mixture was analyzed by a gel retardation assay.Figure S4. (a) Filter-binding assay: The mentioned peptides were bound to α[32P]UTP-labeled HCV SLIV RNA followed by filter-binding assay.Figure S5. Modeled structure of C15 peptide (coloured in cyan) with the interaction between the side chain of Ser 138 with main chain carbonyl oxygen at Leu 135 shown.Figure S6. (a–b) Semiquantitative RT-PCR: Huh7 cells were trans-fected with Rep2a RNA.Figure S7. (a) In vivo labeling: Huh 7 cells were transfected with replicon 2a RNA followed by peptide transduction.Figure S8. (a) Western blot showing the entrapment of FITC-NS3 peptide in virosome.Figure S9. Strategy of in vivo delivery of peptide entrapped virosome in mice.Figure S10. In vivo peptide delivery experiment was performed as described in Supplementary Figure S9.

AcKnoWledGMentsWe thank Dr Stanley Lemon, Dr Takaji Wakita, Dr Charles M Rice, Dr Ralf Bartenschlager, Dr Akio Nomoto for plasmid constructs. We ac-knowledge Dr Eric Gowans for valuable suggestions, comments, and critical reading of the manuscript. We thank our lab members for their helpful discussion. This work was supported by grant from the Department of Biotechnology, Government of India to S.D. U.R and A.K. were supported by predoctoral fellowship from CSIR, India.

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