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Hepatitis C virus NS5A protein modulates template selection by the RNA polymerase in in vitro system
FEBS Letters 583 (2009) 277–280
journal homepage: www.FEBSLetters.org
Hepatitis C virus NS5A protein modulates template selection by the RNA polymerase in in vitro system Alexander V. Ivanov a,b,*, Vera L. Tunitskaya a, Olga N. Ivanova a, Vladimir A. Mitkevich a,b, Vladimir S. Prassolov a, Alexander A. Makarov a, Marina K. Kukhanova a, Sergey N. Kochetkov a a b
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov street, 32, Moscow 119991, Russia University of Oslo, Centre for Medical Study in Russia, Moscow 119334, Russia
a r t i c l e
i n f o
Article history: Received 30 July 2008 Revised 27 November 2008 Accepted 5 December 2008 Available online 13 December 2008 Edited by Hans-Dieter Klenk Keywords: Hepatitis RNA polymerase NS5A protein Phosphorylation
a b s t r a c t Hepatitis C virus (HCV) NS5A phosphoprotein is a component of virus replicase. Here we demonstrate that in vitro unphosphorylated NS5A protein inhibits HCV RNA-dependent RNA polymerase (RdRp) activity in polyA–oligoU system but has little effect on synthesis of viral RNA. The phosphorylated casein kinase (CK) II NS5A protein causes the opposite effect on RdRp in each of these systems. The phosphorylation of NS5A protein with CKII does not affect its affinity to the HCV RdRp and RNA. The NS5A phosphorylation with CKI does not change the RdRp activity. Herein we report evidence that the NS5A prevents template binding to the RdRp. Structured summary: MINT-6803697: CKI (uniprotkb:P97633) phosphorylates (MI:0217) NS5A (uniprotkb:P26662) by protein kinase assay (MI:0424) MINT-6803713: CKII (uniprotkb:P67870) phosphorylates (MI:0217) NS5A (uniprotkb:P26662) by protein kinase assay (MI:0424) Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Hepatitis C virus (HCV) infection is one of the most widespread and dangerous human diseases [1]. The HCV replication complex is composed of viral non-structural proteins (NS3, NS4A, NS4B, NS5A, and NS5B) and several cellular accessory proteins [2]. Many of the replicase components, and NS5A in particular, can modulate numerous cellular processes. The NS5A protein is responsible for viral resistance to interferon a and is involved in altering several signal transduction pathways [3]. A direct interaction of recombinant NS5A protein with viral NS5B protein encoding RNA-dependent RNA polymerase (RdRp) [4,5] and with viral RNA [6] supports the hypothesis that NS5A plays an important role in virus replication. NS5A protein is presented in eukaryotic cells in both an unphosphorylated [6] and two phosphorylated forms (basal p56 and hyperphosphorylated p58) [7]. Basal NS5A phosphorylation affects serine residues [8] in the central and C-terminal regions [7]. Casein kinase (CK) II phosphorylates the C-terminal region [9], whereas
the central region is phosphorylated by an unidentified protein kinase, which is likely to be proline-directed kinase [8]. Currently it is thought that basal phosphorylation of NS5A C-terminal region has no effect on HCV replication [6], however, the effect of basal NS5A phosphorylation on the protein interaction with HCV RdRp is unknown. Herein, we report the in vitro effects of unphosphorylated and CKI- and CKII-phosphorylated NS5A protein on the function of HCV RdRp in different systems. We have demonstrated that unphosphorylated NS5A inhibits polyA-dependent polyU synthesis due to the prevention of the enzyme–template interaction, while the CKII-phosphorylated NS5A form did not affect the RdRp activity in this system. In contrast, when viral RNA was used as a template, the CKII-phosphorylated NS5A protein completely blocked RdRp activity whereas the unphosphorylated NS5A had no effect. In addition, the phosphorylation of NS5A with CKI did not alter RdRp activity in either system. 2. Methods
Abbreviations: HCV, hepatitis C virus; RdRp, RNA-dependent RNA polymerase; CK, casein kinase; UTR, untranslated region * Corresponding author. Address: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, MODFAS, Vavilov Strasse, 32, Moscow 119991, Russia. Fax: +7 499 1351405. E-mail address: [email protected] (A.V. Ivanov).
2.1. Materials CKI (10 U/ll) from rat liver was purchased from Promega. Both C-terminally His-tagged full-length (1–447) NS5A protein and
0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.12.016
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NS5BD55 truncated form of RdRp were expressed and purified as described previously [10]. The N-terminal fragment (1–378) of NS5A protein was purified as described for the full-length NS5A protein [10]. The a-catalytic subunit of CKII from human testis was expressed with N-terminal His-tag from two-cistron vector in E. coli similar to N-terminally His-tagged NS5A and purified under native conditions as described for NS5B protein. Oligonucleotides were obtained from Syntol (Moscow, Russia). [a-32P]UTP and [c-32P]ATP (5000 Ci/mmol) were from Izotop (Russia). All other reagents of highest grade were obtained from Sigma (St. Louis, MI), and the enzymes were purchased from Sybenzyme (Novosibirsk, Russia). (-)IRES was obtained from plasmid pCVH77T by amplification of the corresponding HCV genome region from plasmid pCVH77T with specific oligonucleotides followed by in vitro transcription as described in [10]. 30 Untranslated region (UTR) was synthesized similarly with oligonucleotides 1 (50 -ACATGATCTGCAGAGAGGCC-30 ) and 2 (50 -AAATAATACGACTCACTATAGGTTGGGGTAAACACTCCG-30 ). 2.2. DNA constructs Plasmid for expression of the N-terminal (1–378) NS5A fragment was obtained by amplification of the corresponding region of the gene from pET-21d-2c-5A plasmid [10] with primers T7 (50 -TAATACGACTCACTATAGGG-30 ) and 3 (50 -ATCTCGAGCTTTGTGGCGAGCTCCG-30 . The product was treated with EcoRI and XhoI endonucleases and cloned into the respective sites of pET-21d-2c vector. Plasmid for expression of CKII was constructed by amplification of the target gene from plasmid pGEM-T-CKII with oligonucleotides 4 (50 -GGAATTCCATATGTCCGGACCCGTGCCAAGC-30 ) and 5 (50 -GATCGGATCCTAGGGCCGTTACTGCTGAG-30 ) and cloning of the product into NdeI and BamHI of pET-15b vector.
2.6. RNA binding assays RNA binding assays were performed with Nitrocelluloze TransBlotÒ and nylon Zeta-ProbeÒ (Bio-Rad) membranes as described in [6]. Briefly, the binding reaction mixtures containing 50 -[32P] labeled 2 nM rU12, and 0–500 nM NS5A protein were kept on ice for 30 min and then filtered through the membranes which were dried on air. The radioactivity was visualized as described above. The dissociation constants were determined as described by Huang et al. [6]. 2.7. Isothermal titration calorimetry The thermodynamic parameters of NS5A protein binding to RdRp were measured using a MicroCal VP-ITC instrument (MicroCal, MA). Experiments were carried out at 25 °C in buffer C (50 mM Tris–HCl, pH 7.5, 300 mM NaCl, 10% glycerol, 0.5 mM EDTA, and 5 mM b-mercaptoethanol) similarly to [12]. Briefly, aliquots (10 ll) of NS5A protein were injected into the 1.42 ml cell containing the NS5B solution. The concentration of NS5A and NS5B proteins ranged from 20 to 30 lM and from 1 to 2 lM, respectively. 2.8. UV cross-linking assay of RNA–protein interaction The reaction mixture (20 ll) containing NS5B protein (200 nM) or/and NS5A protein (200 nM), 100 nM 50 -labeled polyA, (-)IRES, or 30 UTR in RdRp activity assay buffer D (20 mM Tris–HCl, pH 7.5, 20 mM KCl, 4 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and 0.1 mg/ml BSA) was kept on ice for 10 min, irradiated at 254 nm for 30 min and directly subjected to 1% agarose gel electrophoresis. The gels were dried and radioactivity was visualized as described above.
2.3. Protein phosphorylation
3. Results and discussion
The reaction mixture contained 0.05–0.4 mg/ml NS5A protein, 0 or 0.05 lCi/ll [c-32P]ATP, 10–100 lM ATP, and 0.01 mg/ml CKII (>100 U/lg) or 0.1 U/ll of CKI, or lysate of Huh7 cell culture (see below) in buffer A (20 mM Tris–HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, and 1 mM DTT). For inhibition of CKII we used 50 lg/ml heparin. After 1 h incubation at 37 °C, the mixtures were analyzed on 10% SDS–PAGE with radioactivity visualization by Packard Cyclone Storage Phosphor System.
3.1. The in vitro phosphorylation of HCV NS5A protein
2.4. Preparation of lysate of Huh7 cell culture Huh7 cells (approximately 3–4 106) were twice washed with PBS, harvested, resuspended and lysed in 200 ll buffer B (25 mM Tris–phosphate, pH 7.8, 2 mM DTT, 10% (v/v) glycerol, 1% (v/v) triton X-100). The resulting lysate was stored on ice and used within 1 h. For phosphorylation reactions, 10 ll of the suspension were added to 1 ml of the reaction mixture. 2.5. The effect of NS5A protein phosphorylation on RdRp activity The NS5B RdRp activity was measured in polyA–oligoU, (-)IRES, or 30 UTR assays as described previously [10,11]. Phosphorylation of NS5A protein with CKI or CKII was conducted in the reaction volume of 170 ll as described in Section 2.3. Aliquots (20 ll) were taken out after 0, 1, 2, 5, 10, 15, 40, and 60 min incubation and kept at 60 °C for additional 10 min for enzyme inactivation. Three ll from each sample were added to RdRp activity assays (17 ll) simultaneously with RNA. The NS5A: NS5B ratio (M/M) was approximately 1:1. To the control mixture an equal amount of buffer A was added. The sample inactivated immediately after kinase addition on ice was referred to as ‘‘unphosphorylated”.
Several research groups have shown that NS5A protein can be phosphorylated in vitro by homogeneous CKII (for example [9]), or protein kinase A [3] to give a basal phosphorylated form p56. The NS5A protein is also a direct substrate of CKIa which can produce both p56 and hyperphosphorylated p58 forms [13]. We examined cellular extracts from Huh7 cells to determine the impact of CKII on NS5A basal phosphorylation and to verify that CKI can participate in basal phosphorylation. Incubation of NS5A protein with [c-32P]ATP and either recombinant CKII or CKI or crude Huh7 cell lysate followed by SDS-PAGE analysis of products revealed that in all cases only p56 was formed (Fig. 1). In the presence of heparin, which is a relatively specific CKII inhibitor [14], the phosphorylation yield for both CKII and Huh7 cell extract was much lower (5.7 and 4.1 fold, respectively), indicating that CKII could be the main protein kinase responsible for basal NS5A phosphorylation (Fig. 1B). During the preparation of this manuscript, similar data were published for another CKII inhibitor TBB [15]. 3.2. Effect of phosphorylated and unphosphorylated NS5A protein on NS5B RdRp activity in the various systems We estimated the effect of the CKI- and CKII-phosphorylated NS5A protein on the enzymatic activity of NS5B protein in polyA–oligoU, (-)IRES, and 30 UTR systems (Fig. 2A–C). The results revealed that unmodified NS5A protein, in a ratio of 1:1 (M/M) to NS5B protein, inhibited polyA-dependent UMP incorporation by 90% (Fig. 2A). With an increase in the CKII phosphorylation level
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Fig. 3. RdRp activity in the presence of NS5A or its truncated form in polyA–oligoU system as a function of the order of components’ addition to the reaction mixture. NS5A was added to the assays after preincubation of RdRp with RNA (lane 1); or simultaneously with RNA (lane 2); or NS5A was preincubated with RdRp before RNA addition (lane 3). The polymerase activity was determined as described in Section 2. Fig. 1. SDS–PAGE analysis of phosphorylation products of NS5A protein or casein by CKI, CKII (A) or Huh7 cell lysate (B) in the absence or presence of heparin.
of NS5A, the inhibitory effect subsided and the exhaustively phosphorylated NS5A protein no longer blocked the activity of NS5B protein. An opposite effect was observed using natural virus (-)IRES and 30 UTR (Fig. 2B); the phosphorylation of NS5A protein by CKI had no effect on NS5B RdRp activity (Fig. 2C). Similar results were observed for both NS5BD21 and NS5BD55 truncated RdRp forms and for N- and C-terminally His-tagged NS5A proteins (Fig. S1). RdRp inhibition by NS5A was non-competitive towards polyA template however a mixed type inhibition was observed with the (-)IRES template (Fig. S2). To understand the mechanism of inhibition, we hypothesized that the NS5A capacity to inhibit the NS5B activity is a function of the order of components’ addition to the assay. As can be seen in Fig. 3, unphosphorylated NS5A protein had no effect on the RdRp activity when added to the assay after preincubation of NS5B protein with an oligoU–polyA primer-template complex (lane 1), whereas the preliminary incubation of NS5A with RdRp before addition of polyA–oligoU resulted in a nearly complete loss of RdRp activity (lane 3). The C-terminally truncated NS5A, which cannot be phosphorylated by CKII, did not inhibit RdRp when added simultaneously with RNA (lane 2). The unphosphorylated NS5A did not affect the RdRp activity in any combination. These data indicate that C-terminus of NS5A protein prevents binding of the template to the polymerase. 3.3. Phosphorylation of NS5A protein does not alter its binding capacity to RNA or NS5B protein but prevents interaction of the NS5B protein with RNA template We compared the Kd values for complexes of unphosphorylated and CKII-phosphorylated NS5A with either RNA or polymerase to
verify if the effects of two NS5A forms upon RdRp activity could be explained by their different affinity to the polymerase or template. Isothermal titration calorimetry revealed that phosphorylation of NS5A by CKII had no impact on the Kd of NS5A–RdRp complexes (Table S1). This result is supported by Shirota et al. [4], who have demonstrated that the NS5B-binding sites (105– 163 and 276–334 a.a.) of the NS5A protein are located outside the C-terminal region (379–447 a.a.). Since NS5A has affinity to polyU/G but not to polyA/C oligonucleotides [6], we determined the Kd for [NS5A–rU12] complex using the filter binding assay. It was found that both NS5A forms showed similar affinity towards rU12 with K0.5 values of 1.84 and 1.24 nM, respectively (Table S2). The direct interaction of RdRp with labeled polyA, (-)IRES, or 30 UTR templates in the absence or presence of NS5A protein was studied by UV cross-linking experiments (Fig. 4). One can see that NS5B, but not NS5A, displays substantial affinity towards either template. Addition of unphosphorylated NS5A protein to the mixture containing polyA and NS5B protein noticeably diminished the amount of the [NS5B–RNA] complex formed (Fig. 4A). At the same time the efficacy of cross-linking in the presence of CKII-phosphorylated NS5A protein was similar to that in the control experiment with the individual RdRp (lane 2 vs. 6). The opposite effect, although less distinct, was observed in the case of (-)IRES and 30 UTR (Fig. 4B and C). Based on these results, we suggested that the unphosphorylated NS5A C-terminus serves as a gate for the binding of artificial polyA template to the NS5B protein, whereas the phosphorylated protein prevents binding with viral RNA. It is also likely that the phosphorylation of NS5A C-terminus changes the conformation and/or location of this polypeptide fragment since it is not involved in NS5A–NS5B interaction. These
Fig. 2. Correlation between phosphorylation degree of NS5A protein and RdRp activity in polyA–oligoU (A), (-)IRES or 30 UTR (B) assays. Effect of phosphorylation degree of NS5A protein by CKI on RdRp activity in polyA–oligoU and (-)IRES systems (C).
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Fig. 4. UV-induced cross-linking of the RdRp with polyA (A), (-)IRES (B) or 30 UTR (C) templates in the absence or presence of unphosphorylated or phosphorylated NS5A protein. The efficiencies of RNA–protein complex formations were quantified with TotalLab software as a ratio of labeled RNA–protein complex to the total amount of labeled RNA in a probe. Asterisk denotes an artifact.
assumptions are supported by the observation that C-terminally truncated NS5A did not alter NS5B interactions with any template. It could also be assumed that polyA- and (-)IRES- binding sites in HCV RdRp structure may not fully overlap, and that the NS5A Cterminal domain of a respective NS5A form is likely to shield one of these sites from interaction wtih an RNA template. We have demonstrated the mechanism of template selection for HCV RdRp by NS5A protein in in vitro assays. However, literature data obtained in the cellular replicon system provide evidence that prevention of the protein phosphorylation by substitution of all serine residues in the C-terminal region has no effect on HCV replication [6]. An apparent inconsistency between results of in vitro and in vivo experiments may be accounted for by many factors. The mechanism of RdRp activity regulation by NS5A protein presented in this manuscript was observed for soluble proteins. However, in infected cells both RdRp and NS5A are localized on ER membrane and lipid raft by their membrane associating domains [16], interact with HCV NS4B protein [17] and cellular vesicle membrane transport protein (hVAP-33) [18]. They also bind other cellular proteins, and all these interactions might either change orientation of NS5A and NS5B or alter position of NS5A C-terminal region thus not allowing the latter to block RdRp. Recently it has been demonstrated that phosphorylation of NS5A C-terminus is required for virus nucleocapsid formation [19]. During this first step of virus particle assembly phosphorylated NS5A allows core protein to bind HCV genome RNA, whereas unphosphorylated protein does not. So, NS5A basal phosphorylation is crucial for other stages of virus life cycle. Acknowledgements The authors are grateful to Dr. N. Gurskaya who kindly provided pGEM-T-CKII plasmid, Dr. Astier-Gin for plasmid pCVH77C, and Prof. K. Seley-Radtke for a critical reading of the manuscript. This work was supported by the Russian Foundation for Basic Research (Projects 08-04-00552 and 07-04-00391), Presidium of Russian Academy of Sciences (Program on Molecular and Cellular Biology), and Federal special program (State Contract No. 02.512.11.2237). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2008.12.016. References [1] Rosen, H.R. and Gretch, D.R. (1999) Hepatitis C virus: current understanding and prospects for future therapies. Mol. Med. Today 5, 393–399.
[2] Waris, G., Sarker, S. and Siddiqui, A. (2004) Two-step affinity purification of the hepatitis C virus ribonucleoprotein complex. RNA 10, 321–329. [3] Macdonald, A. and Harris, M. (2004) Hepatitis C virus NS5A: tales of a promiscuous protein. J. Gen. Virol. 85, 2485–2502. [4] Shirota, Y., Luo, H., Qin, W., Kaneko, S., Yamashita, T., Kobayashi, K. and Murakami, S. (2002) Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity. J. Biol. Chem. 277, 11149–11155. [5] Huang, L., Sineva, E.V., Hargittai, M.R., Sharma, S.D., Suthar, M., Raney, K.D. and Cameron, C.E. (2004) Purification and characterization of hepatitis C virus nonstructural protein 5A expressed in Escherichia coli. Protein Expr. Purif. 37, 144– 153. [6] Huang, L., Hwang, J., Sharma, S.D., Hargittai, M.R., Chen, Y., Arnold, J.J., Raney, K.D. and Cameron, C.E. (2005) Hepatitis C virus nonstructural protein 5A (NS5A) is an RNA-binding protein. J. Biol. Chem. 280, 36417–36428. [7] Tanji, Y., Kaneko, T., Satoh, S. and Shimotohno, K. (1995) Phosphorylation of hepatitis C virus-encoded nonstructural protein NS5A. J. Virol. 69, 3980–3986. [8] Reed, K.E., Xu, J. and Rice, C.M. (1997) Phosphorylation of the hepatitis C virus NS5A protein in vitro and in vivo: properties of the NS5A-associated kinase. J. Virol. 71, 7187–7197. [9] Kim, J., Lee, D. and Choe, J. (1999) Hepatitis C virus NS5A protein is phosphorylated by casein kinase II. Biochem. Biophys. Res. Commun. 257, 777–781. [10] Ivanov, A.V., Korovina, A.N., Tunitskaya, V.L., Kostyuk, D.A., Rechinsky, V.O., Kukhanova, M.K. and Kochetkov, S.N. (2006) Development of the system ensuring a high-level expression of hepatitis C virus nonstructural NS5B and NS5A proteins. Protein Expr. Purif. 48, 14–23. [11] Reigadas, S., Ventura, M., Sarih-Cottin, L., Castroviejo, M., Litvak, S. and AstierGin, T. (2001) HCV RNA-dependent RNA polymerase replicates in vitro the 30 terminal region of the minus-strand viral RNA more efficiently than the 30 terminal region of the plus RNA. Eur. J. Biochem. 268, 5857–5867. [12] Mitkevich, V.A., Kononenko, A.V., Petrushanko, I.Y., Yanvarev, D.V., Makarov, A.A. and Kisselev, L.L. (2006) Termination of translation in eukaryotes is mediated by the quaternary eRF1eRF3GTPMg2+ complex. The biological roles of eRF3 and prokaryotic RF3 are profoundly distinct. Nucleic Acids Res. 34, 3947–3954. [13] Quintavalle, M., Sambucini, S., Summa, V., Orsatti, L., Talamo, F., De Francesco, R. and Neddermann, P. (2007) Hepatitis C virus NS5A is a direct substrate of casein kinase I-{alpha}, a cellular kinase identified by inhibitor affinity chromatography using specific NS5A hyperphosphorylation inhibitors. J. Biol. Chem. 282, 5536–5544. [14] Hathaway, G.M., Lubben, T.H. and Traugh, J.A. (1980) Inhibition of casein kinase II by heparin. J. Biol. Chem. 255, 8038–8041. [15] Dal Pero, F., Di Maira, G., Marin, O., Bortoletto, G., Pinna, L.A., Alberti, A., Ruzzene, M. and Gerotto, M. (2007) Heterogeneity of CK2 phosphorylation sites in the NS5A protein of different hepatitis C virus genotypes. J. Hepatol. 47, 768–776. [16] Gosert, R., Jendrsczok, W., Berke, J.M., Brass, V., Blum, H.E. and Moradpour, D. (2005) Characterization of nonstructural protein membrane anchor deletion mutants expressed in the context of the hepatitis C virus polyprotein. J. Virol. 79, 7911–7917. [17] Dimitrova, M., Imbert, I., Kieny, M.P. and Schuster, C. (2003) Protein–protein interactions between hepatitis C virus nonstructural proteins. J. Virol. 77, 5401–5414. [18] Gao, L., Aizaki, H., He, J.W. and Lai, M.M. (2004) Interactions between viral nonstructural proteins and host protein hVAP-33 mediate the formation of hepatitis C virus RNA replication complex on lipid raft. J. Virol. 78, 3480–3488. [19] Masaki, T., Suzuki, R., Murakami, K., Aizaki, H., Ishii, K., Murayama, A., Date, T., Matsuura, Y., Miyamura, T., Wakita, T. and Suzuki, T. (2008) Interaction of hepatitis C virus nonstructural protein 5A with core protein is critical for the production of infectious virus particles. J. Virol. 82, 7964–7976.
journal homepage: www.FEBSLetters.org
Hepatitis C virus NS5A protein modulates template selection by the RNA polymerase in in vitro system Alexander V. Ivanov a,b,*, Vera L. Tunitskaya a, Olga N. Ivanova a, Vladimir A. Mitkevich a,b, Vladimir S. Prassolov a, Alexander A. Makarov a, Marina K. Kukhanova a, Sergey N. Kochetkov a a b
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov street, 32, Moscow 119991, Russia University of Oslo, Centre for Medical Study in Russia, Moscow 119334, Russia
a r t i c l e
i n f o
Article history: Received 30 July 2008 Revised 27 November 2008 Accepted 5 December 2008 Available online 13 December 2008 Edited by Hans-Dieter Klenk Keywords: Hepatitis RNA polymerase NS5A protein Phosphorylation
a b s t r a c t Hepatitis C virus (HCV) NS5A phosphoprotein is a component of virus replicase. Here we demonstrate that in vitro unphosphorylated NS5A protein inhibits HCV RNA-dependent RNA polymerase (RdRp) activity in polyA–oligoU system but has little effect on synthesis of viral RNA. The phosphorylated casein kinase (CK) II NS5A protein causes the opposite effect on RdRp in each of these systems. The phosphorylation of NS5A protein with CKII does not affect its affinity to the HCV RdRp and RNA. The NS5A phosphorylation with CKI does not change the RdRp activity. Herein we report evidence that the NS5A prevents template binding to the RdRp. Structured summary: MINT-6803697: CKI (uniprotkb:P97633) phosphorylates (MI:0217) NS5A (uniprotkb:P26662) by protein kinase assay (MI:0424) MINT-6803713: CKII (uniprotkb:P67870) phosphorylates (MI:0217) NS5A (uniprotkb:P26662) by protein kinase assay (MI:0424) Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Hepatitis C virus (HCV) infection is one of the most widespread and dangerous human diseases [1]. The HCV replication complex is composed of viral non-structural proteins (NS3, NS4A, NS4B, NS5A, and NS5B) and several cellular accessory proteins [2]. Many of the replicase components, and NS5A in particular, can modulate numerous cellular processes. The NS5A protein is responsible for viral resistance to interferon a and is involved in altering several signal transduction pathways [3]. A direct interaction of recombinant NS5A protein with viral NS5B protein encoding RNA-dependent RNA polymerase (RdRp) [4,5] and with viral RNA [6] supports the hypothesis that NS5A plays an important role in virus replication. NS5A protein is presented in eukaryotic cells in both an unphosphorylated [6] and two phosphorylated forms (basal p56 and hyperphosphorylated p58) [7]. Basal NS5A phosphorylation affects serine residues [8] in the central and C-terminal regions [7]. Casein kinase (CK) II phosphorylates the C-terminal region [9], whereas
the central region is phosphorylated by an unidentified protein kinase, which is likely to be proline-directed kinase [8]. Currently it is thought that basal phosphorylation of NS5A C-terminal region has no effect on HCV replication [6], however, the effect of basal NS5A phosphorylation on the protein interaction with HCV RdRp is unknown. Herein, we report the in vitro effects of unphosphorylated and CKI- and CKII-phosphorylated NS5A protein on the function of HCV RdRp in different systems. We have demonstrated that unphosphorylated NS5A inhibits polyA-dependent polyU synthesis due to the prevention of the enzyme–template interaction, while the CKII-phosphorylated NS5A form did not affect the RdRp activity in this system. In contrast, when viral RNA was used as a template, the CKII-phosphorylated NS5A protein completely blocked RdRp activity whereas the unphosphorylated NS5A had no effect. In addition, the phosphorylation of NS5A with CKI did not alter RdRp activity in either system. 2. Methods
Abbreviations: HCV, hepatitis C virus; RdRp, RNA-dependent RNA polymerase; CK, casein kinase; UTR, untranslated region * Corresponding author. Address: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, MODFAS, Vavilov Strasse, 32, Moscow 119991, Russia. Fax: +7 499 1351405. E-mail address: [email protected] (A.V. Ivanov).
2.1. Materials CKI (10 U/ll) from rat liver was purchased from Promega. Both C-terminally His-tagged full-length (1–447) NS5A protein and
0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.12.016
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NS5BD55 truncated form of RdRp were expressed and purified as described previously [10]. The N-terminal fragment (1–378) of NS5A protein was purified as described for the full-length NS5A protein [10]. The a-catalytic subunit of CKII from human testis was expressed with N-terminal His-tag from two-cistron vector in E. coli similar to N-terminally His-tagged NS5A and purified under native conditions as described for NS5B protein. Oligonucleotides were obtained from Syntol (Moscow, Russia). [a-32P]UTP and [c-32P]ATP (5000 Ci/mmol) were from Izotop (Russia). All other reagents of highest grade were obtained from Sigma (St. Louis, MI), and the enzymes were purchased from Sybenzyme (Novosibirsk, Russia). (-)IRES was obtained from plasmid pCVH77T by amplification of the corresponding HCV genome region from plasmid pCVH77T with specific oligonucleotides followed by in vitro transcription as described in [10]. 30 Untranslated region (UTR) was synthesized similarly with oligonucleotides 1 (50 -ACATGATCTGCAGAGAGGCC-30 ) and 2 (50 -AAATAATACGACTCACTATAGGTTGGGGTAAACACTCCG-30 ). 2.2. DNA constructs Plasmid for expression of the N-terminal (1–378) NS5A fragment was obtained by amplification of the corresponding region of the gene from pET-21d-2c-5A plasmid [10] with primers T7 (50 -TAATACGACTCACTATAGGG-30 ) and 3 (50 -ATCTCGAGCTTTGTGGCGAGCTCCG-30 . The product was treated with EcoRI and XhoI endonucleases and cloned into the respective sites of pET-21d-2c vector. Plasmid for expression of CKII was constructed by amplification of the target gene from plasmid pGEM-T-CKII with oligonucleotides 4 (50 -GGAATTCCATATGTCCGGACCCGTGCCAAGC-30 ) and 5 (50 -GATCGGATCCTAGGGCCGTTACTGCTGAG-30 ) and cloning of the product into NdeI and BamHI of pET-15b vector.
2.6. RNA binding assays RNA binding assays were performed with Nitrocelluloze TransBlotÒ and nylon Zeta-ProbeÒ (Bio-Rad) membranes as described in [6]. Briefly, the binding reaction mixtures containing 50 -[32P] labeled 2 nM rU12, and 0–500 nM NS5A protein were kept on ice for 30 min and then filtered through the membranes which were dried on air. The radioactivity was visualized as described above. The dissociation constants were determined as described by Huang et al. [6]. 2.7. Isothermal titration calorimetry The thermodynamic parameters of NS5A protein binding to RdRp were measured using a MicroCal VP-ITC instrument (MicroCal, MA). Experiments were carried out at 25 °C in buffer C (50 mM Tris–HCl, pH 7.5, 300 mM NaCl, 10% glycerol, 0.5 mM EDTA, and 5 mM b-mercaptoethanol) similarly to [12]. Briefly, aliquots (10 ll) of NS5A protein were injected into the 1.42 ml cell containing the NS5B solution. The concentration of NS5A and NS5B proteins ranged from 20 to 30 lM and from 1 to 2 lM, respectively. 2.8. UV cross-linking assay of RNA–protein interaction The reaction mixture (20 ll) containing NS5B protein (200 nM) or/and NS5A protein (200 nM), 100 nM 50 -labeled polyA, (-)IRES, or 30 UTR in RdRp activity assay buffer D (20 mM Tris–HCl, pH 7.5, 20 mM KCl, 4 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and 0.1 mg/ml BSA) was kept on ice for 10 min, irradiated at 254 nm for 30 min and directly subjected to 1% agarose gel electrophoresis. The gels were dried and radioactivity was visualized as described above.
2.3. Protein phosphorylation
3. Results and discussion
The reaction mixture contained 0.05–0.4 mg/ml NS5A protein, 0 or 0.05 lCi/ll [c-32P]ATP, 10–100 lM ATP, and 0.01 mg/ml CKII (>100 U/lg) or 0.1 U/ll of CKI, or lysate of Huh7 cell culture (see below) in buffer A (20 mM Tris–HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, and 1 mM DTT). For inhibition of CKII we used 50 lg/ml heparin. After 1 h incubation at 37 °C, the mixtures were analyzed on 10% SDS–PAGE with radioactivity visualization by Packard Cyclone Storage Phosphor System.
3.1. The in vitro phosphorylation of HCV NS5A protein
2.4. Preparation of lysate of Huh7 cell culture Huh7 cells (approximately 3–4 106) were twice washed with PBS, harvested, resuspended and lysed in 200 ll buffer B (25 mM Tris–phosphate, pH 7.8, 2 mM DTT, 10% (v/v) glycerol, 1% (v/v) triton X-100). The resulting lysate was stored on ice and used within 1 h. For phosphorylation reactions, 10 ll of the suspension were added to 1 ml of the reaction mixture. 2.5. The effect of NS5A protein phosphorylation on RdRp activity The NS5B RdRp activity was measured in polyA–oligoU, (-)IRES, or 30 UTR assays as described previously [10,11]. Phosphorylation of NS5A protein with CKI or CKII was conducted in the reaction volume of 170 ll as described in Section 2.3. Aliquots (20 ll) were taken out after 0, 1, 2, 5, 10, 15, 40, and 60 min incubation and kept at 60 °C for additional 10 min for enzyme inactivation. Three ll from each sample were added to RdRp activity assays (17 ll) simultaneously with RNA. The NS5A: NS5B ratio (M/M) was approximately 1:1. To the control mixture an equal amount of buffer A was added. The sample inactivated immediately after kinase addition on ice was referred to as ‘‘unphosphorylated”.
Several research groups have shown that NS5A protein can be phosphorylated in vitro by homogeneous CKII (for example [9]), or protein kinase A [3] to give a basal phosphorylated form p56. The NS5A protein is also a direct substrate of CKIa which can produce both p56 and hyperphosphorylated p58 forms [13]. We examined cellular extracts from Huh7 cells to determine the impact of CKII on NS5A basal phosphorylation and to verify that CKI can participate in basal phosphorylation. Incubation of NS5A protein with [c-32P]ATP and either recombinant CKII or CKI or crude Huh7 cell lysate followed by SDS-PAGE analysis of products revealed that in all cases only p56 was formed (Fig. 1). In the presence of heparin, which is a relatively specific CKII inhibitor [14], the phosphorylation yield for both CKII and Huh7 cell extract was much lower (5.7 and 4.1 fold, respectively), indicating that CKII could be the main protein kinase responsible for basal NS5A phosphorylation (Fig. 1B). During the preparation of this manuscript, similar data were published for another CKII inhibitor TBB [15]. 3.2. Effect of phosphorylated and unphosphorylated NS5A protein on NS5B RdRp activity in the various systems We estimated the effect of the CKI- and CKII-phosphorylated NS5A protein on the enzymatic activity of NS5B protein in polyA–oligoU, (-)IRES, and 30 UTR systems (Fig. 2A–C). The results revealed that unmodified NS5A protein, in a ratio of 1:1 (M/M) to NS5B protein, inhibited polyA-dependent UMP incorporation by 90% (Fig. 2A). With an increase in the CKII phosphorylation level
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Fig. 3. RdRp activity in the presence of NS5A or its truncated form in polyA–oligoU system as a function of the order of components’ addition to the reaction mixture. NS5A was added to the assays after preincubation of RdRp with RNA (lane 1); or simultaneously with RNA (lane 2); or NS5A was preincubated with RdRp before RNA addition (lane 3). The polymerase activity was determined as described in Section 2. Fig. 1. SDS–PAGE analysis of phosphorylation products of NS5A protein or casein by CKI, CKII (A) or Huh7 cell lysate (B) in the absence or presence of heparin.
of NS5A, the inhibitory effect subsided and the exhaustively phosphorylated NS5A protein no longer blocked the activity of NS5B protein. An opposite effect was observed using natural virus (-)IRES and 30 UTR (Fig. 2B); the phosphorylation of NS5A protein by CKI had no effect on NS5B RdRp activity (Fig. 2C). Similar results were observed for both NS5BD21 and NS5BD55 truncated RdRp forms and for N- and C-terminally His-tagged NS5A proteins (Fig. S1). RdRp inhibition by NS5A was non-competitive towards polyA template however a mixed type inhibition was observed with the (-)IRES template (Fig. S2). To understand the mechanism of inhibition, we hypothesized that the NS5A capacity to inhibit the NS5B activity is a function of the order of components’ addition to the assay. As can be seen in Fig. 3, unphosphorylated NS5A protein had no effect on the RdRp activity when added to the assay after preincubation of NS5B protein with an oligoU–polyA primer-template complex (lane 1), whereas the preliminary incubation of NS5A with RdRp before addition of polyA–oligoU resulted in a nearly complete loss of RdRp activity (lane 3). The C-terminally truncated NS5A, which cannot be phosphorylated by CKII, did not inhibit RdRp when added simultaneously with RNA (lane 2). The unphosphorylated NS5A did not affect the RdRp activity in any combination. These data indicate that C-terminus of NS5A protein prevents binding of the template to the polymerase. 3.3. Phosphorylation of NS5A protein does not alter its binding capacity to RNA or NS5B protein but prevents interaction of the NS5B protein with RNA template We compared the Kd values for complexes of unphosphorylated and CKII-phosphorylated NS5A with either RNA or polymerase to
verify if the effects of two NS5A forms upon RdRp activity could be explained by their different affinity to the polymerase or template. Isothermal titration calorimetry revealed that phosphorylation of NS5A by CKII had no impact on the Kd of NS5A–RdRp complexes (Table S1). This result is supported by Shirota et al. [4], who have demonstrated that the NS5B-binding sites (105– 163 and 276–334 a.a.) of the NS5A protein are located outside the C-terminal region (379–447 a.a.). Since NS5A has affinity to polyU/G but not to polyA/C oligonucleotides [6], we determined the Kd for [NS5A–rU12] complex using the filter binding assay. It was found that both NS5A forms showed similar affinity towards rU12 with K0.5 values of 1.84 and 1.24 nM, respectively (Table S2). The direct interaction of RdRp with labeled polyA, (-)IRES, or 30 UTR templates in the absence or presence of NS5A protein was studied by UV cross-linking experiments (Fig. 4). One can see that NS5B, but not NS5A, displays substantial affinity towards either template. Addition of unphosphorylated NS5A protein to the mixture containing polyA and NS5B protein noticeably diminished the amount of the [NS5B–RNA] complex formed (Fig. 4A). At the same time the efficacy of cross-linking in the presence of CKII-phosphorylated NS5A protein was similar to that in the control experiment with the individual RdRp (lane 2 vs. 6). The opposite effect, although less distinct, was observed in the case of (-)IRES and 30 UTR (Fig. 4B and C). Based on these results, we suggested that the unphosphorylated NS5A C-terminus serves as a gate for the binding of artificial polyA template to the NS5B protein, whereas the phosphorylated protein prevents binding with viral RNA. It is also likely that the phosphorylation of NS5A C-terminus changes the conformation and/or location of this polypeptide fragment since it is not involved in NS5A–NS5B interaction. These
Fig. 2. Correlation between phosphorylation degree of NS5A protein and RdRp activity in polyA–oligoU (A), (-)IRES or 30 UTR (B) assays. Effect of phosphorylation degree of NS5A protein by CKI on RdRp activity in polyA–oligoU and (-)IRES systems (C).
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Fig. 4. UV-induced cross-linking of the RdRp with polyA (A), (-)IRES (B) or 30 UTR (C) templates in the absence or presence of unphosphorylated or phosphorylated NS5A protein. The efficiencies of RNA–protein complex formations were quantified with TotalLab software as a ratio of labeled RNA–protein complex to the total amount of labeled RNA in a probe. Asterisk denotes an artifact.
assumptions are supported by the observation that C-terminally truncated NS5A did not alter NS5B interactions with any template. It could also be assumed that polyA- and (-)IRES- binding sites in HCV RdRp structure may not fully overlap, and that the NS5A Cterminal domain of a respective NS5A form is likely to shield one of these sites from interaction wtih an RNA template. We have demonstrated the mechanism of template selection for HCV RdRp by NS5A protein in in vitro assays. However, literature data obtained in the cellular replicon system provide evidence that prevention of the protein phosphorylation by substitution of all serine residues in the C-terminal region has no effect on HCV replication [6]. An apparent inconsistency between results of in vitro and in vivo experiments may be accounted for by many factors. The mechanism of RdRp activity regulation by NS5A protein presented in this manuscript was observed for soluble proteins. However, in infected cells both RdRp and NS5A are localized on ER membrane and lipid raft by their membrane associating domains [16], interact with HCV NS4B protein [17] and cellular vesicle membrane transport protein (hVAP-33) [18]. They also bind other cellular proteins, and all these interactions might either change orientation of NS5A and NS5B or alter position of NS5A C-terminal region thus not allowing the latter to block RdRp. Recently it has been demonstrated that phosphorylation of NS5A C-terminus is required for virus nucleocapsid formation [19]. During this first step of virus particle assembly phosphorylated NS5A allows core protein to bind HCV genome RNA, whereas unphosphorylated protein does not. So, NS5A basal phosphorylation is crucial for other stages of virus life cycle. Acknowledgements The authors are grateful to Dr. N. Gurskaya who kindly provided pGEM-T-CKII plasmid, Dr. Astier-Gin for plasmid pCVH77C, and Prof. K. Seley-Radtke for a critical reading of the manuscript. This work was supported by the Russian Foundation for Basic Research (Projects 08-04-00552 and 07-04-00391), Presidium of Russian Academy of Sciences (Program on Molecular and Cellular Biology), and Federal special program (State Contract No. 02.512.11.2237). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2008.12.016. References [1] Rosen, H.R. and Gretch, D.R. (1999) Hepatitis C virus: current understanding and prospects for future therapies. Mol. Med. Today 5, 393–399.
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