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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Hepatitis C virus (HCV) is a leading cause of chronic liver disease worldwide and establishes a persistent infection in more than 60% of infected individuals. This high frequency of persistent infection indicates that HCV has evolved efficient strategies to interfere with the adaptive and innate immune response and to occupy and use host cell infrastructure. The present study provides evidence that c-Src, a member of the Src family kinases that participates in many signal transduction pathways, represents an essential host factor exploited for viral replication. c-Src directly interacts with the viral RNA-dependent RNA polymerase (NS5B) via its SH3 domain and with the nonstructural phosphoprotein NS5A via its SH2 domain. Both interactions are required to maintain the protein–protein interaction of NS5A and NS5B, which has been previously demonstrated to be essential for viral replication. Accordingly, HCV genome replication and production of the viral proteins was strongly reduced upon small interfering RNA–mediated knockdown of c-Src or in the presence of the tyrosine kinase inhibitor herbimycin A. This effect could not be rescued by supplementation of the two other ubiquitously expressed Src family kinases Fyn or Yes. Conclusion: Our data suggest that c-Src participates in the formation of an NS5A/NS5B protein complex that is required for efficient replication of HCV. (HEPATOLOGY 2011;53:-)

Hepatitis C virus (HCV) is a global health burden and is a major cause for chronic liver disease leading to liver cirrhosis and subsequent complications, such as portal hypertension and hepatocellular carcinoma.1 For reasons that are not well understood, persistent infection will develop in over 60% of infected individuals. The virus thus must have evolved strategies to subvert the host antiviral defense, to temper the inflammatory response, to prevent the virus-infected cell from undergoing apoptosis, and to use host cell infrastructure without major cytopathogenicity.

The 9.6-kb, positive strand RNA of HCV encodes a large open reading frame flanked by highly structured untranslated regions at the 5′ and 3′ end. The translation of the open reading frame results in a precursor polyprotein that is co- and posttranslationally processed into three structural proteins and seven nonstructural proteins, termed the hydrophobic peptide p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B, of which the nonstructural proteins NS3 through NS5B are essentially required for autonomous replication.2 NS3/4A is a virus-encoded protease/helicase, whereas NS5A is a phosphoprotein with multiple functions and NS5B is the RNA-dependent RNA polymerase representing the core unit of the viral replication complex. Over recent decades, it has become increasingly evident that many HCV proteins interfere with components of signaling cascades of the host cell, thereby influencing cell growth, apoptosis, antiviral responses, release of inflammatory mediators, and other functions of the host cell.

Our knowledge of host proteins and signaling cascades essential for viral replication and the capacity of HCV to influence and use the respective signaling pathways is fragmentary. Host factors that have been shown to be essential for HCV replication include cyclophilin A, heat shock protein 90 (Hsp90), the vesicle-associated membrane protein–associated proteins A and B, and the protein kinase Akt. Knockdown of the expression of these genes or application of inhibitors such as cyclosporin A to inhibit cyclophilin A, geldanamycin to antagonize Hsp90 activity,3 or triciribine to suppress the constitutive Akt activity observed in the presence of HCV4 resulted in impaired HCV replication (reviewed in Bode et al.5).

Apart from this, members of the Src protein tyrosine kinase family (SFK) have been reported to be important for viral replication or production and release of infectious particles of different viruses, such as human immunodeficiency virus or hepatitis B virus.5 SFKs mediate intracellular signals of many different cellular receptors that control a diverse spectrum of biological activities. This kinase family consists of eight members—namely Lyn, Hck, Lck, Blk, c-Src, Fyn, Yes, and Fgr—that show similar modes of regulation but differ with respect to cell type specificity and function. Thus, Src, Fyn, and Yes are expressed in most tissues, whereas the other SFK members are primarily found in hematopoietic cells, with the exception of Lck and Lyn, which have also been detected in neurons (reviewed in Parsons and Parsons6 and Okutani et al.7).

Although there are some conflicting reports indicating that NS5A interacts with SFK members and that they may influence viral replication,8, 9 the role of SFKs for HCV replication and the interaction of HCV with SFKs are not well understood.

The present study addresses the interrelationship between HCV and c-Src and provides evidence that c-Src is important for complex formation of NS5A and NS5B—a complex known to be required for viral RNA replication.10

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Materials.

The antibody specific for c-Src was purchased from Millipore (Schwalbach, Germany), for glutathione S-transferase (GST) from Cell Signaling (Danvers, MA), for NS3 from Abcam (Cambridge, UK), and for NS5A and NS5B were obtained from Alexis (San Diego, CA). The c-Src inhibitor herbimycin A was purchased from Calbiochem (Schwalbach, Germany).

Cell Culture.

The human hepatoma cells Huh 7 wild-type and Huh 7.5 as well as the Huh 5-15, Huh 9-13, and Huh 11-7 cell lines harboring the HCV replicase complex2 were cultivated in Dulbecco's modified Eagle's medium/nutrient mix F-12 (Invitrogen, Karlsruhe, Germany) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (Perbio, Bonn, Germany). Medium was changed 16 hours before experiments were performed.

Plasmids.

The complementary DNA for human c-Src was amplified from complementary DNA generated from messenger RNA of Huh 7 cells and subcloned into the p3XFlag-CMV-7.1 expression vector (Sigma, Saint Louis, MO). Glutathione-S-transferase (GST) fusion proteins were generated by subcloning using the pGEX-6P-3 expression vector system from Amersham (Freiburg, Germany). The full-length GST-c-Src and GST-NS5B construct, as well as the deletion mutants GST-src-ΔSH3 (deletion of aa 51-148), GST-src-ΔSH2 (deletion of aa 164-243), GST-src-ΔSH1 (deletion of aa 247-536), GST-src-SH1 (deletion of aa 1-252), GST-NS5B-Δ1-357, GST-NS5B-Δ382-591, and GST-NS5B-Δ402-591, were generated using standard cloning procedures as mentioned above.

Real-Time Polymerase Chain Reaction.

Real-time polymerase chain reaction (PCR) was performed as described.4 The primers used are listed in the Supporting Information. Specificity of real-time PCR was controlled by no template and no reverse-transcriptase controls. Semiquantitative PCR results were obtained using the ΔCT method and threshold values were normalized to hnSDHA.

Transfection Procedure and Small Interfering RNA.

Huh cells were transiently transfected using c-Src–specific small interfering RNA (siRNA) from Thermo Scientific Dharmacon (Lafayette, CO) according to the manufacturer's instructions or a Lipofectamine 2000–based protocol, which is outlined in the Supporting Information for self-designed siRNA (sequences are listed in the Supporting Information).

Western Blot Analysis.

At the end of experimental treatment, cells were washed twice with phosphate-buffered saline (PBS) supplemented with 0.1 mM Na3VO4, solubilized in lysis buffer (see Supporting Information), and sonicated 2 times for 20 seconds at 4°C. Protein concentration was estimated by using the BioRad protein assay. Equal amounts of protein were subjected to western blot analysis.

Infection of Huh7.5 Cells.

Persistent infection of Huh7.5 cells was established by infection of cells with HCV strain JC111, 12 24 hours after seeding with a multiplicity of infection of 1. Cells were subsequently subjected to repetitive cycles of passaging and used after 2 weeks, which corresponds to four passages.

Expression and Purification of GST Proteins.

BL21 Escherichia coli bacteria (Promega) were transformed with the respective expression vector and subsequently grown in 2YT medium with 50 μg/mL ampicillin, until an optical density of 1.5 at 600 nm was reached. Thereafter, GST fusion protein expression was induced by adding 0.1 mM isopropyl-beta-D-thiogalactopyranoside, and incubation was continued for another 4 hours at 30°C. The bacteria were then pelletized at 4°C for 10 minutes at 7,700g and resuspended in 10 mL PBS containing Complete Protease Inhibitor Cocktail (Roche). After sonication, Triton X-100 was added to a final concentration of 1% (vol/vol) and incubated for 30 minutes at 4°C. The suspension was centrifuged at 4°C for 10 minutes at 12,000g and the supernatant, containing the GST proteins, stored at −20°C, and used for pull-down assays.

GST Pull-Down.

GST pull-down was performed as outlined in the Supporting Information by incubating glutathion-sepharose beads loaded with the respective GST fusion proteins with Huh 9-13 protein extracts. Co-precipitated proteins were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (12% gel) and subsequent immunoblot analysis.

Immunoprecipitation.

For co-immunoprecipitation, 4 μg of specific antibody and 500 μg of Huh 9-13 protein lysate were rotated for 2 hours at 4°C in a total volume of 500 μL PBS. Afterward, 2× washed protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA) was added and incubated overnight at 4°C. Thereafter, the samples were washed 3 times with Triton-wash-buffer, resuspended and denatured in 2× SDS-PAGE buffer for 5 minutes at 95°C, supplemented at 4°C for 1 minute at 13.000 rpm and subjected to SDS-PAGE (12% gel).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

c-Src Activity Is Required for Viral Replication.

It has been recently reported that the benzoquinoid ansamycin antibiotic herbimycin A, which is known to inhibit the activity of viral Src and c-Src,13-16 affects HCV replication. However, due to the propinquity of herbimycin A to geldanamycin, which binds to Hsp90 and alters its function, this finding has been linked to a potential role of Hsp90 for HCV replication, but not to a possible role of c-Src for HCV replication.3 Indeed, as shown in Fig. 1, herbimycin A concentration-dependently almost completely abrogates HCV replication when analyzed in the subgenomic replicon cell lines Huh 9-132 (Fig. 1), Huh 5-15, and Huh 11-7 (Supporting Information Fig. 1) with maximal inhibitory effects after an incubation period of 72 hours, depending on the concentration used. In line with these observations, treatment with 0.1 μM herbimycin A also strongly suppressed HCV replication in Huh7.5 cells persistently infected with the JC1 strain (Fig. 1D). In contrast to HCV, replication of vaccinia virus or vesicular stomatitis virus, both of which replicate as HCV at extranuclear sites in the cytoplasm in Huh7 cells, was resistant to treatment with 0.1 μM herbimycin A. This finding suggests that the effect of herbimycin A on HCV replication is indeed specific rather than an unselective impairment of the host cell resulting in a curtailed efficiency of viral replication (Supporting Information Fig. 2).

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Figure 1. Inhibition of Src activity suppresses HCV replication. (A-C) Hepatoma cell lines harboring the HCV genotype 1–derived subgenomic replicon (Huh 9-13) were pretreated with herbimycin A at the concentrations indicated or with dimethyl sulfoxide (DMSO) as a control. After the respective time periods, the indicated total RNA (A,B) or protein extracts (C) were prepared as outlined in Materials and Methods. Total RNA was subjected to semiquantitative real-time PCR for the abundance of HCV RNA. Semiquantitative PCR results were obtained using the ΔCT method. HCV RNA was normalized to succinate dehydrogenase complex subunit A (SDHA), and relative HCV RNA levels were expressed as fractions of the normalized value of the wild-type control, which was set to 100%. Data are presented as the mean + SD. (D) persistent infection of Huh 7.5 cell lines with the JC1 strain of HCV was established as outlined in Materials and Methods. Subsequently, 3 × 106 cells were seeded into 6-well plates and treated with herbimycin A or DMSO for control as indicated 24 hours after seeding. Protein extracts were prepared after the respective time periods. For C) and D) 30 μg of total protein lysates were separated by SDS-PAGE using a 10% separation gel and subsequently analyzed by immunoblot using antibodies specifically recognizing NS3, NS5A, or NS5B as depicted. Expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was analyzed using a GAPDH-specific antibody and was used as a loading control.

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To assess whether the inhibition of HCV replication by herbimycin A might be due to its inhibitory effect on c-Src, three different siRNAs directed against c-Src were established and used to suppress c-Src expression and compared with GFP siRNA as a control (Fig. 2A,B). In addition, as a second approach, a commercially available c-Src siRNA pool and respective nontarget control siRNA (Dharmacon) was used (Fig. 2C,D). As shown in Fig. 2, down-regulation of c-Src expression was achieved by all siRNAs used and resulted in an effective decrease of the abundance of HCV RNA (Fig. 2A) and protein (Fig. 2B,C). Thereby suppression of viral RNA and protein expression closely coincided with the degree c-Src expression was suppressed. It can be concluded from these data that c-Src is required for the maintenance of sufficient replication and that the effect of herbimycin A on HCV replication may be indeed due to inhibition of c-Src activity. This view is further supported by the observation that siRNA-mediated suppression of c-Src expression by 71 ± 4% lowered the half maximal inhibitory concentration (IC50) of herbimycin A to a similar extent from 0.11 μM to 0.038 μM (Fig. 2D). This inhibition of HCV replication upon suppression of c-Src expression by specific siRNA could be rescued by expression of neither Yes nor Fyn (Supporting Information Fig. 3). Thus, the two other ubiquitously expressed Src family members Yes and Fyn are not able to substitute c-Src. According to this, knockdown of Yes and Fyn by siRNA did not largely affect viral protein expression (Supporting Information Fig. 4). In summary, these data suggest that, from those Src family members that are ubiquitously expressed, c-Src plays a relevant role for HCV replication, whereas Fyn and Yes seem to be dispensable.

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Figure 2. HCV replication requires c-Src expression. (A,B) Huh 9-13 cells were transfected with siRNA I, II, or III or a combination established to specifically suppress c-Src expression or with GFP siRNA for control as described in Materials and Methods. (C) Huh 9-13 cells were transfected with different concentrations or a constant amount of 50 nM at different time points with a pool of siRNA specifically directed against c-Src or scrambled siRNA. Total RNA extracts (A) were prepared and subjected to real-time PCR for the abundance of HCV RNA, c-Src messenger RNA, or succinate dehydrogenase complex subunit A (SDHA) messenger RNA using respective primer pairs. Semiquantitative PCR results were calculated as outlined in Fig. 1 and are presented as the mean + SD. Total protein extracts were prepared and analyzed for the expression of NS3, NS5A, NS5B, and c-Src via immunoblotting subsequent to separation of 30 μg of total protein via SDS-PAGE with a 12% separation gel. (D) cells were transfected with 50 nM of siRNA specifically directed against c-Src or with scrambled siRNA for control as described above. Twenty-four hours thereafter, cells were treated with different concentrations of herbimycin A or with DMSO for control for another 48 hours. After this time period, total protein extracts were prepared and 40 μg of protein was analyzed for NS5B, c-Src, and GAPDH expression via western blot analysis. Blots were scanned and digitally processed using the Kodak station 4000 MM and the chemi-imager software Total Lab 100 from Nonlinear Dynamics (Durham, NC). Signals from NS5B were normalized to GAPDH signals and are presented as percent of control, which was set to 100%. Percentages were plotted against the concentration of herbimycin A employed; linear regression lines were calculated, and the IC50 of herbimycin A was deduced from the respective regression lines. Data are presented as the mean ± SD of at least three independent experiments.

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The SH3 Domain of c-Src Is Required for its Interaction with NS5B, Whereas the SH2 Domain Is Essential for its Interaction with NS5A.

Because herbimycin A and c-Src siRNAs significantly affected the abundance of viral genomic RNA, we raised the question of whether c-Src binds to the viral RNA-dependent RNA polymerase (NS5B). As shown in Fig. 3A, NS5B could be coprecipitated with c-Src–specific antibodies from whole protein extracts prepared from Huh 9-13 cells harboring the subgenomic HCV replicon. Accordingly, in pull-down assays using GST-tagged c-Src, NS5B could also be precipitated from cell lysates prepared from replicon-expressing Huh 9-13 cell lines (Fig. 3B) or from Huh cell lines infected with two different JFH1-derived viral HCV strains (Supporting Information Fig. 5). Conversely, GST-tagged NS5B was also able to precipitate c-Src (Fig. 4). Apart from confirming the assumption that NS5B interacts with c-Src, the pull-down assays using GST-tagged c-Src further indicated that NS5A also binds to c-Src. These data suggest that either a protein complex comprising c-Src, NS5A, and NS5B is formed or two independent complexes comprising c-Src plus NS5A or c-Src plus NS5B (Figs. 3 and 4).

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Figure 3. c-Src interacts with NS5A via its SH2 domain and its SH3 domain is required for interaction with NS5B. (A) Total protein extracts were subjected to immunoprecipitation using antibodies specific for c-Src. Subsequently, the precipitated proteins were separated via SDS-PAGE using a 12% gel, and the abundance of the precipitated c-Src and the coprecipitated NS5B was determined via immunoblotting using antibodies that specifically recognize c-Src or NS5B. (B) Full-length c-Src or respective deletion mutants of c-Src fused to GST were generated as depicted and expressed in BL21 Escherichia coli and coupled to glutathione-conjugated sepharose beads as outlined in Materials and Methods. After extensive rinsing, the respective GST fusion protein–loaded beads were incubated with total protein extracts prepared from untreated Huh 9-13 cells and subsequently separated via SDS-PAGE as outlined in Materials and Methods. The interaction of NS5A or NS5B with the respective GST fusion proteins was analyzed via immunoblotting using antibodies specifically raised against NS5A or NS5B. GST-specific antibodies were used as a loading control.

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Figure 4. NS5B interacts with c-Src via a region comprising a prolin-rich motif. (A) Two prolin-rich motives, potentially responsible for SH3 domain interactions, could be identified within the HCV genotype 1b, isolate Con1 sequence (CAB46913) using SH3-Hunter software. Sequences highlighted in grey boxes were depicted as motif 1 (M1) or motif 2 (M2). (B) Full-length NS5B or the depicted deletion mutants of NS5B fused to GST were generated and coupled to glutathione-conjugated sepharose beads as outlined in Materials and Methods. Subsequently, the GST fusion protein–loaded beads were incubated with 500 μg of total protein extracts prepared from untreated Huh 9-13 cells, and after the respective washing steps, the resulting precipitates were separated via SDS-PAGE as outlined in Materials and Methods. The interaction of c-Src with the respective GST fusion proteins was analyzed via immunoblotting using antibodies specifically raised against c-Src. GST-specific antibodies were used as a loading control.

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To define the regions of c-Src that are required for the interaction with NS5A and NS5B in more detail, GST-tagged deletion mutants of c-Src were constructed and used for pull-down assays. As demonstrated in Fig. 3B, c-Src deletion mutants lacking the SH3 domain were unable to coprecipitate NS5B, whereas coprecipitation of NS5A was reduced but not abrogated. In contrast, deletion of the SH2 domain completely interrupted the interaction of c-Src with NS5A, but did not affect the interaction with NS5B. This indicates that the interaction of c-Src with NS5A requires the SH2 domain, whereas the interaction with NS5B depends on the presence of the SH3 domain.

Pull-down assays using isolated GST-tagged SH3 domains of c-Src, Fyn, Hck, Lck, and c-Abl (Fig. 5) further indicate that the interactions of NS5B with the SH3 domain of c-Src is substantially stronger than those of NS5B with the isolated SH3 domains of the other SFK members tested (Fyn, Hck, Lck) or of the tyrosine-kinase c-Abl, which was rather faint (Fyn, Hck, Lck) or not detectable (c-Abl).

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Figure 5. NS5B interacts with isolated GST-tagged SH3 domains of depicted SFK members. Isolated SH3 domains of Src, Fyn, Hck, Lck, and Abl fused to GST were generated as indicated, and the interaction of NS5B with the respective SH3 domains of the different Src family kinase members was analyzed via pull-down assay as described in Figs. 4 and 5. The precipitated proteins were separated via SDS-PAGE using a 12% separation gel, and the coprecipitated NS5B was analyzed via immunoblotting using an antibody specifically raised against NS5B. A GST-specific antibody was used as a loading control.

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Sequence analysis of NS5B for the prevalence of proline-rich motifs that represent putative sites for the interaction of NS5B with the SH3 domain of c-Src revealed two possible binding sites (Fig. 4) located between aa 347 and 355 (termed M1) and between aa 385 and 392 (termed M2). Deletion of the C-terminal, M2 comprising part of NS5B (deletion of aa 382 to 591), but not of the N-terminal part of NS5B that contains M1 (deletion of aa 1-357) strongly reduces the interaction between NS5B and c-Src (Fig. 4), which was not the case when deletion of the C-terminal part did not include motif 2 (deletion of aa 402-591). This suggests that the M2-containing region located between aa 382-402 is important for the interaction of NS5B with c-Src. The fact that deletion of the N-terminal part of NS5B completely abrogates the interaction with NS5A but did not affect the interaction of NS5B with c-Src indicates that irrespective of their interaction with c-Src, NS5A and NS5B also directly interact with each other (Fig. 4). The latter observation indicates that, although c-Src undergoes complex formation with NS5A and/or NS5B either as one ternary complex or as two independent complexes, the interaction of NS5A and NS5B also involves direct protein–protein interactions, which is in line with previous reports.17

Herbimycin A Blocks the Interaction of NS5A and NS5B.

It has been reported that NS5A and NS5B directly interact with each other and that this complex formation of NS5A and NS5B is essentially required for efficient viral replication.10, 17 Because the data presented herein suggest that c-Src is part of this protein complex, the question was addressed whether the interaction of NS5A and NS5B is sensitive toward the tyrosine kinase inhibitor herbimycin A. As shown in Fig. 6A, treatment of Huh 9-13 cells harboring the subgenomic replicon of HCV with herbimycin A for 14 hours results in a substantial reduction of the amount of NS5B coprecipitated with NS5A, suggesting that the interaction of NS5A and NS5B is sensitive to herbimycin A.

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Figure 6. Herbimycin A treatment or c-Src knockdown by siRNA disrupts the interaction between NS5A and NS5B. (A) Huh 9-13 cells were treated with 0.1 μM herbimycin A or DMSO for an incubation period of 14 hours as depicted. (B) Huh 9-13 cells were treated with 50 nM c-Src specific siRNA or a control mix for 36 hours. Thereafter, total protein extracts were prepared and 500 μg of protein were subjected to immunoprecipitation using antibodies specific for NS5A. Subsequently, the precipitated proteins were analyzed via SDS-PAGE using a 12% separation gel. The abundance of the coprecipitated NS5B was determined via immunoblotting using an NS5B-specific antibody. As an input control, 10 μg of the respective extracts were separated on the same gel and analyzed via immunoblotting using antibodies specific for NS5A and NS5B.

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That complex formation of NS5A and NS5B indeed requires the presence of c-Src is further substantiated by the fact that suppression of c-Src expression using specific siRNA likewise resulted in an impaired protein–protein interaction of NS5A and NS5B if analyzed by co-immunoprecipitation experiments using antibodies specifically directed against NS5A (Fig. 6B). It can be concluded from these data that c-Src is required to enhance complex formation of NS5A and NS5B, which in turn is essential for viral replication.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Replication of viruses completely relies on host cell infrastructure, and therefore viruses have evolved mechanisms to control and use cellular machineries. As shown in the present study, HCV appears to exploit the cellular tyrosine kinase c-Src to achieve efficient RNA replication.

SFKs are enzymes of key importance for the control of a diverse spectrum of biological activities (reviewed in Parsons and Parsons6). c-Src, the cellular prototype of this kinase family, has been originally discovered as the mammalian homologue of viral Src kinase encoded by the Rous sarcoma virus.18, 19 c-Src is ubiquitously expressed and is of particular importance for governing cellular processes associated with cellular proliferation, differentiation, and cell survival such as cell cycle control, protein synthesis, organization of the cytoskeleton, and the cell adhesion network.6, 7

The present study provides evidence that c-Src contributes toward maintenance of HCV replication, as suppression of c-Src expression by specific siRNAs resulted in an effective down-regulation of HCV replication (Fig. 2). Neither Fyn nor Yes was able to annihilate this inhibitory effect of c-Src knockdown on HCV replication. This suggests that c-Src plays a specific role for HCV replication and cannot be substituted by the two other ubiquitously expressed SFK members Yes and Fyn, a notion that is further supported by the fact that siRNA directed against these two kinases has no influence on HCV replication. In line with this, HCV replication is also highly sensitive toward the protein tyrosine kinase inhibitor herbimycin A (Fig. 1), which has been originally described as an inhibitor of viral Src activity14 and subsequently demonstrated to likewise inhibit c-Src activity.13, 15 Our notion that this effect of herbimycin A on HCV replication is indeed mainly due to the inhibition of c-Src function is further supported by the observation that down-regulation of c-Src expression by siRNA is accompanied by a reduction of the IC50 of herbimycin A, which is commensurate to the reduction of c-Src protein levels (Fig. 2D).

It has been demonstrated in previous reports that HCV-encoded proteins interact with members of the Src family kinases. Notably, NS5A has been suggested to interact with the SH3 domain of Hck, Lck, Lyn, and Fyn, but interestingly not with that of c-Src.8, 20 The interaction of NS5A with the respective member of the SFK family was suggested to inhibit the activity of Hck, Lck, and Lyn and enhances activation of Fyn, which in turn resulted in an increased activation of STAT3.8 In contrast to this, a recent report used an siRNA-based screening approach and identified the C-terminal Src kinase, which mediates phosphorylation of the C-terminal inhibitory tyrosine residue of SFKs, to be required for replication. This effect of C-terminal Src kinase was suggested to be due to negative regulation of Fyn,9 because siRNA-mediated suppression of Fyn expression was reported to enhance replication, whereas siRNAs directed against the other ubiquitously expressed SFKs c-Src and Yes were reported to have no effect on replication. In the present study, we were unable to confirm the proposed inhibitory effect of Fyn on HCV replication.9 Instead, substantial evidence is provided that functional c-Src is important for HCV replication and that lack of c-Src cannot be substituted by the other two ubiquitously expressed SFK members Yes and Fyn (Supporting Information Fig. 3). The assumption that SFK activity provides supportive rather than inhibitory effects on HCV replication is further supported by the observation that treatment with two SFK inhibitors SU6656 or PP2 likewise impairs HCV replication (Supporting Information Fig. 6).

Previous data from our group indicate that HCV does not influence the phosphorylation of c-Src at the regulatory tyrosine residues 418 and 529,4 suggesting that HCV uses c-Src without affecting its phosphorylation state. Indeed, the data provided herein suggest that complex formation of c-Src with the virus-encoded proteins NS5A and NS5B (Figs. 3-5 and Supporting Information Fig. 2) is important. This protein–protein interaction between NS5B and c-Src requires the SH3 domain of c-Src (Fig. 3B) and a region of NS5B located between aa 382 and 402 (Fig. 4B), whereas the SH2 domain of c-Src is important for the interaction between NS5A and c-Src (Fig. 3B).

The finding that the interaction of NS5A and c-Src mainly requires a functional SH2 domain of c-Src, whereas the SH3 domain is of less importance, was surprising, because NS5A contains a highly conserved C-terminal polyproline motif with the consensus sequence Pro-X-X-Pro-X-Arg. This motif represents a canonical SH3 domain binding site required for the interaction with the SH3 domains of a variety of cellular proteins, including the SFK members Hck, Lck, Fyn, and Lyn, but interestingly not c-Src.8, 21 The exact nature of the interaction between NS5A and the SH2 domain of c-Src is not clear and needs to be further analyzed. It is known that protein–SH2 domain interactions depend on phosphorylation of tyrosine residues within the SH2 domain–interacting region of the respective protein. NS5A is a highly phosphorylated protein that has been demonstrated to be phosphorylated at several serine residues, but also comprises several tyrosine residues. This makes the identification of the tyrosine residue that is involved in the interaction with c-Src a challenging task, which will be the goal of our future work. However, the observation that the interaction of NS5A and NS5B is sensitive toward herbimycin A (Fig. 6A) points toward a role of tyrosine kinase activity for the complex formation of NS5A and NS5B. In this context, it is important to note that complex formation of NS5A and NS5B has been demonstrated to be crucial for viral replication.10

Whereas NS5A seems to mainly interact with the SH2 domain of c-Src, the interaction of c-Src and NS5B requires the SH3 domain of c-Src (Fig. 3). Therefore, NS5B does not exclusively interact with the SH3 domain of c-Src, but also associates with the isolated SH3 domains of other SFK members such as Fyn, Hck, and Lck, although to a much weaker extent (Fig. 5). The relevance of the latter observation remains to be established. In particular, only Fyn is thought to be ubiquitously expressed, whereas expression of Lck and Hck is reported to be restricted to hematopoietic and in part neuronal cell types.6, 7 As mentioned above, neither Fyn nor Yes, which apart from c-Src and Fyn are also present in most cell types, is able to substitute c-Src function for replication (Supporting Information Figs. 3 and 4). Hence, from those SFK members known to be ubiquitously expressed, only c-Src seems to be suitable for the requirements of HCV, and it is likely that its ability to simultaneously interact with NS5B and NS5A and to enhance complex formation of NS5A and NS5B is of major importance. Apart from this, it should be noted that the SH3 domains from Hck, Lck, Lyn, and Fyn have been reported to have a high affinity to NS5A, whereas the SH3 domain of c-Src does not interact with NS5A.8, 20, 21 It is therefore conceivable that, in contrast to the SH3 domain of c-Src, the observed interaction of the SH3 domains of Hck, Lck, Lyn, and Fyn with NS5B might be also due to an indirect, NS5A-mediated interaction with NS5B that has been demonstrated to directly interact with NS5A.10, 17 This direct interaction of NS5A and NS5B involves two reported discontinuous regions within NS5A10, 17 and is strongly enhanced by c-Src (Fig. 6B), which becomes recruited to the NS5A–NS5B protein complex (Figs. 3 and 4). Recruitment of NS5A to this protein complex requires, apart from the SH2 domain of c-Src (Fig. 3), the N-terminal part of NS5B (Fig. 4), suggesting that the direct interaction of NS5A and NS5B might be important for the effect of c-Src on NS5A–NS5B complex formation.

In conclusion, these data point toward an important role of c-Src for viral replication. The data suggest that c-Src supports HCV replication by enhancing complex formation between NS5A and NS5B, which has been demonstrated to be required for HCV replication.10, 17 c-Src forms a complex comprising NS5A and NS5B by recruiting NS5A via its SH2 domain and the viral RNA–dependent RNA polymerase NS5B via its SH3 domain. This presumably enhances the direct interaction of NS5A and NS5B, which, apart from two discontinuous regions identified within NS5A,17 may also depend on the N-terminal part of NS5B (Fig. 4). The exact nature of the resulting protein complex and in particular the respective motifs within c-Src, NS5A, and NS5B that mediate complex formation has to be further clarified in order to specifically interfere with the formation of this complex.

Prevention of complex formation between c-Src, NS5A, and NS5B, for example by the use of small molecules, may represent a potential therapeutic strategy to impair viral replication. In contrast to therapeutical targeting of HCV-encoded proteins, such as inhibition of the viral protease or the viral polymerase, a strategy that targets virus–host interactions required for sufficient virus replication or survival of the virus would have the advantage of a comparably high genetic barrier and should therefore be less susceptible toward development of viral resistance.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Anja Voges for excellent technical assistance.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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