- Email: [email protected]
Classical swine fever virus NS3 enhances RNA-dependent RNA polymerase activity by binding to NS5B
Virus Research 148 (2010) 17–23
Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Classical swine fever virus NS3 enhances RNA-dependent RNA polymerase activity by binding to NS5B Ping Wang, Yujing Wang, Yu Zhao, Zailing Zhu, Jialin Yu, Lingzhu Wan, Jun Chen, Ming Xiao ∗ Research Institute for Agricultural Microbiology, College of Life and Environment Sciences, Shanghai Normal University, Shanghai, 200234, China
a r t i c l e
i n f o
Article history: Received 12 September 2009 Received in revised form 24 November 2009 Accepted 25 November 2009 Available online 29 November 2009 Keywords: Classical swine fever virus NS3 NS5B NS3–NS5B interaction
a b s t r a c t NS3 of pestiviruses contains a protease domain and a RNA helicase/NTPase domain. Contradictory results have been reported regarding NS3 in RNA synthesis. To investigate the effect of NS3 on classical swine fever virus (CSFV) NS5B RNA-dependent RNA polymerase activity (RdRp) activity and NS3–NS5B interaction, RdRp reactions, GST-pull-down assays and co-immunoprecipitation analyses containing NS5B and either of NS3 protein and the different truncated NS3 mutants were performed, respectively. We found that NS3 stimulated NS5B RdRp activity in a dose-dependent manner by binding to NS5 through a NS3 protease domain. Furthermore, mapping important regions of the NS3 protease domain was carried out by deletion mutagenesis, associated with RdRp reactions, GST-pull-down assays and coimmunoprecipitation analyses. Results showed that stimulation of CSFV NS5B RdRp activity was obtained by NS3 binding to NS5B through a 31-amino acid fragment at the N-terminal end of NS3 protease domain, which mediated a specific NS3–NS5B interaction. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Classical swine fever virus (CSFV) is a small enveloped virus, which is an important pathogen of pigs, and often causes major losses in stock farming. CSFV is a member of the genus Pestivirus which also comprises bovine viral diarrhea virus 1 (BVDV-1), BVDV2, and border disease virus (BDV) (Becher and Thiel, 2002; Heinz et al., 2000). The genus Pestivirus belongs to the family Flaviviridae. The hepatitis C virus (HCV) also belongs to this family (Cuthbert, 1994). CSFV genome is a single plus-strand RNA, and contains a single large open reading frame (ORF), a 5 untranslated region (5 UTR) and a 3 untranslated region (3 UTR). The ORF encodes a polyprotein of approximately 3900 amino acids. The viral genome enters host cell and here is translated into the polyprotein, which is processed by viral and cellular proteases into 12 mature proteins (4 structural proteins and 8 nonstructural proteins): Npro , P70, NS2, NS3, NS4A, NS4B, NS5A, NS5B (Moennig and Plagemann, 1992). The CSFV NS5B protein has an RNA-dependent RNA polymerase (RdRp) activity (Steffen et al., 1999; Xiao et al., 2002, 2006), contains a conserved GDD motif necessary for the catalytic activity (Wang et al., 2007), transcribes the genome into minus strands, which serve as templates to produce more plusstrand RNAs for packaging into progeny viral capsids (Gong et al., 1996). During RNA synthesis, replicative intermediate and replica-
∗ Corresponding author. Tel.: +86 21 64321022; fax: +86 21 65642468. E-mail address: [email protected] (M. Xiao). 0168-1702/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2009.11.015
tive forms have been detected (Gong et al., 1996). Recent evidence has indicated that NS3 protein is also important in viral replication (Gu et al., 2000; Kolykhalov et al., 2000; Piccininni et al., 2002; Sheng et al., 2007). NS3 is a multifunctional protein possessing serine protease, RNA helicase, and nucleoside triphosphatase (NTPase) activities located in two functionally distinct domains. The N-terminal one-third of pestiviral NS3 primarily serves as a protease to process the viral polyprotein (Xu et al., 1997a). The helicase and NTPase activities are localized to the C-terminal of NS3 protein (Suzich et al., 1993; Warrener and Collett, 1995). It has been reported that NS5A protein is an essential component of the viral RNA replication machinery and may also function in modulation of the host cell environment (Masaki et al., 2008; Tellinghuisen et al., 2004, 2006). In addition, NS4B and NS4A are vital for viral replication (Lindströma et al., 2006; Lundin et al., 2003; Moulin et al., 2007). However, interaction of these proteins and the effects of those interactions on viral replication remain poorly understood. In this paper, we investigated the NS3–NS5B interaction and the effect of this interaction on CSFV RdRp activity by deletion mutagenesis of NS3 protein. 2. Materials and methods 2.1. Protein expression and purification Prokaryotic expression of CSFV NS5B protein, NS3 and its truncated forms were performed as described previously (Sheng et al., 2007; Xiao et al., 2006). The cDNAs encoding full-length NS3 (NS3F) (amino acids 1590–2272), truncated NS3 protease
18
P. Wang et al. / Virus Research 148 (2010) 17–23
(NS3P) (amino acids 1590–1763), truncated NS3 helicase (NS3H) (amino acids 1764–2272), NS3delN1–20 (amino acids 1610–2272), NS3delN1–40, (amino acids 1630–2272), NS3delN1–70 (amino acids 1660–2272), and NS5B (amino acids 3181–3898) were obtained by RT-PCR from the genome of CSFV Shimen strain, and cloned into pET28(a) vector, respectively. Expression of NS3delN40–70 and NS3HadN40–70 were obtained by deletion of amino acids 40–70 and by deletion of amino acids 1–39 and 71–174 from N-terminal end of NS3 (Fig. 4). A His6 tag was added to the C terminus of each of these proteins to facilitate protein. The inserted regions of all clones were sequenced through dideoxynucleotide sequencing and no changes were found. These resulting plasmids were introduced into the Escherichia coli strain BL21(DE3) for expression driven by T7 RNA polymerase. Expression was induced by addition of isopropylthiogalactoside (IPTG). The bacterial cell culture was harvested by centrifugation at 6000 × g for 10 min, and washed in phosphate-buffered saline (PBS). The cells from 1000 ml were resuspended in 20 ml of the buffer containing 50 mM Na-phosphate [pH 8.0], 300 mM NaCl, 10 mM imidazole, 10 mM -mercaptoethanol, 10% glycerol, 1% Nonidet P-40, supplemented with 1 mM phenylmethylsulfonyl fluoride and 10 mM leupeptin. Cells were lysed by freezing and thawing, followed by sonication. The cleared lysates were obtained by centrifugation at 35,000 × g for 15 min, and then, purified using nickel–nitrilotriacetic acid (Ni–NTA)–sepharose resin (Gibco BRL) following the manufacturer’s direction. These CSFV proteins were collected and dialyzed in the buffer A (50 mM Tris–HCl [pH 8.0], 1 mM DTT, 50 mM NaCl, 5 mM MgCl2 , 10% glycerol), respectively. These protein solutions and dilutions of bovine serum albumin with known concentration were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%). The gels with the samples were stained with Coomassie brilliant blue. The amount of each of these proteins was determined by densitometry scanning and comparing the two samples on the same gel. Purified proteins were separated by SDS-PAGE. To express a GST-NS5B fusion protein, the fragments encoding NS5B (amino acids 3181–3898) were amplified by PCR, engineered with a BamHI site and an XhoI site. PCR products were digested with BamHI/XhoI and inserted into a pGEX-4T-1 vector (Amersham Pharmacia Biotech), in frame with the GST-encoding sequence, to generate pGEX-4T/NS5B. The GST or GST-NS5B fusion protein was expressed in Escherichia coli BL21(DE3), purified with glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) according to the manufacturer’s instructions, as described previously (Dimitrova et al., 2003). In brief, expression was induced by addition of IPTG. The cells from 500 ml of culture were then harvested and sonicated in 45 ml of PBS buffer supplemented with a protease inhibitor cocktail and 1% Triton X-100. Insoluble materials were pelleted at 15,000 × g for 10 min, and 500 l of a 50% slurry of glutathione-Sepharose 4B beads was added to the clarified supernatants. The beads were allowed to bind proteins for 1 h, washed three times in PBS, and finally resuspended in 500 l of PBS supplemented with 1% Triton X-100. 2.2. RdRp assays RdRp assays were performed essentially as described previously (Xiao et al., 2004). Total volume was 50 ml, containing the following supplements: 50 mM Hepes (pH 8.0), 5 mM MgCl2 , 10 M DTT, 25 mM KCl, l mM EDTA, 20U RNasin, 50 g actinomycin D (Sigma), 200 M each NTP, 1 l of RNA template (250 ng/ml), 100 ng NS5B proteins and increasing amounts of NS3F, NS3H or NS3P (0, 100, 150, 200, 250,300, and 350 ng, respectively). The mixture was incubated at 37 ◦ C for 1 h, and the reaction was stopped by the addition of 2 l of EDTA (200 mM). The reaction samples were extracted with phenol/chloroform, and RNAs were precipitated with isopropyl alcohol.
2.3. Quantification of viral RNA Quantification of viral RNA was determined by real-time RT-PCR. Forward primer (nt 12071–12093, 5 GCGCGGGTAACCCGGGATCTGAA-3 ), reverse primer (nt 12174–12190, 5 -CAGTTCTTACTCATTCA-3 ), and TaqMan probe (nt 12103–12124, FAM-5 -AGGACCCTATTGTAGATAACAC-3 -TAMRA) were designed based on the 3 UTR sequence of CSFV Shimen strain (accession number: AF092448). Total RNA was extracted from products of RdRp assays with phenol/chloroform. After ethanol precipitation, the RNA was dried, and redissolved in 20 l of double distilled H2 O. The concentration of RNA was determined by measuring its absorbance at 260 nm. RT was performed using Superscript II reverse transcriptase (Invitrogen). The quantitative real-time PCR was run using the AbiPrism7000 sequence detection system (Applied Biosystems). PCR were performed for 40 cycles with cycling conditions of 15 s at 95 ◦ C and 1 min at 60 ◦ C. In vitro-transcribed CSFV RNA of known concentrations were run in parallel reactions, used to generate a standard curve. 2.4. Cell culture PK-15 cells, the natural host of CSFV were used for expressing the CSFV NS5B protein. They were obtained from the China Center for Type Culture Collection, Wuhan. The cells were cultured in DMEM (Gibco BRL) supplemented with 10% of fetal calf serum, penicillin (100 U/ml) and streptomycin (100 g/ml) at 37 ◦ C in a humidified 5% CO2 atmosphere. 2.5. GST-pull-down assay Approximately 2 g of GST or GST-NS5B fusion protein immobilized on 20 l of GST resin was preblocked with 1% bovine serum albumin and then incubated with 2 g of NS3 wild type or mutants in 300 l of PBST buffer (phosphate-buffered saline containing 1% Triton X-100) for 3 h on a rotating device at room temperature. After washing with PBST, the bound proteins were fractionated by SDSPAGE (10%) and subjected to Western blot analysis with anti-NS3 monoclonal antibody. 2.6. Co-immunoprecipitation analysis For co-immunoprecipitation analyses, the sequences encoding NS3F, NS3H, NS3P, NS3delN1–20, NS3delN1–40, NS3delN1–70, NS3delN40–70, and NS3HadN40–70 were obtained by PCR from the pET28(a) vectors containing these protein-encoding regions used for above prokaryotic expression, engineered with BamHI and XbaI sites and cloned into a pcDNA3.1/N-FLAG vector. CSFV NS5B (amino acids 3181–3898) was cloned into pcDNA3.1 vector as described previously (Xiao et al., 2003). The resulting recombinant vectors were used for co-transfection of PK-15 cells, respectively. PK-15 cells transfected with the above plasmids were harvested, washed and sonicated in RIPA buffer consisting of 150 mM NaCl, 0.5% Triton X-100, 10 mM Triton-HCl (pH 7.5). Lysates were centrifuged at 12,000 × g for 10 min at 4 ◦ C and the supernatants was immunoprecipitated with appropriate antibodies for 1 h at 4 ◦ C, followed by incubation with 20 l protein G-agarose beads (Pharmacia) at 4 ◦ C for 1 h. After washing with RIPA buffer, the bound proteins were eluted, fractionated by SDS-PAGE (10%), transferred onto nitrocellulose membranes, and subjected to Western blot analysis with anti-FLAG monoclonal antibody or anti-NS5B antibody. Protein bands were visualized by enhanced chemiluminescence according to the manufacturer’s instructions (Amersham Pharmacia Biotech).
P. Wang et al. / Virus Research 148 (2010) 17–23
19
3. Results 3.1. Stimulation of NS5B RdRp activity by NS3 To examine the effects of NS3 on CSFV NS5B polymerase activity, we expressed the viral NS5B protein, full-length NS3 protein (NS3F), the truncated NS3 protein with postulated helicase domain (NS3H) and another truncated NS3 protein (NS3H) with NS3 protease (NS3P). The NS5B RdRp activities were tested in the presence of increasing amount of NS3F, NS3H and NS3P, respectively. The 3 UTR of CSFV Shimen strain genome was served as a template. To exclude the possibility that the terminal nucleotidyl transferase activity of CSFV NS5B influences the value for its RdRp activity, we used a 3 UTR with oxidized 3 -terminal hydroxyl group (Xiao et al., 2004, 2006). Quantification of synthesized RNA was determined by using real-time RT-PCR. To normalize data, the amount of CSFV RNA in each sample was divided by the amount detected in the NS5B RdRp assay in the absence of NS3 and its truncated forms under same conditions. The experiment was repeated three times. As shown in Fig. 1, NS3F stimulated RdRp activity, and the stimulation was in agreement with increasing amount of purified NS3F, while NS3H and NS3P did not increase NS5B RdRp activity. These results suggested that stimulation of NS5B RdRp activity by NS3 protein needed NS3 helicase domain and NS3 protease domain, and there might be an interaction between NS5B and NS3, NS3 helicase or NS3 protease domain. 3.2. NS3–NS5B interaction by NS3 protease domain To elucidate whether NS5B interacts with NS3 or its truncated forms, a GST-pull-down assay was performed. NS3F, NS3H or NS3P was mixed with the CSFV NS5B bound to glutathione-agarose, and GST protein alone was served as a control. Pulled down proteins were fractioned by SDS-PAGE and subjected to Western blot analysis. Results showed that NS3F was pulled down with GST-NS5B, not with GST alone (Fig. 2, lanes 2 and 1), indicating a specific NS3–NS5B interaction. It was found that NS3P was pulled down with GST-NS5B, but NS3H was not (lanes 4 and 3), implying that the NS3 protease domain mediated the NS3–NS5B interaction, and the NS3 helicase domain did not. To determine whether the interaction of NS5B and either of NS3 and its truncated forms occurs in the cell, co-immunoprecipitation analyses were performed. PK15 cells were transiently cotransfected with the pcDNA3.1 vector containing NS5B and either of the pcDNA3.1/N-FLAG vectors containing NS3 and its truncated forms. Coprecipitated proteins were detected by Western blot analysis. A specific interaction between the NS5B and either of NS3F and NS3P was observed, indicating that CSFV NS5B interacted with either of the full-length NS3 and the NS3 protease domain in PK-15 cells (Fig. 3). These results strongly suggested that stimulation of CSFV NS5B RdRp activity was obtained by NS3 binding to NS5B, and the NS5B-binding site on NS3 might be located in the NS3 protease domain. 3.3. Influence of deletion NS3 mutants on NS5B RdRp activity To determine which region of NS3 protease domain is important to stimulation of NS5B RdRp activity, deletion mutagenesis of NS3 protease domain was carried out. A series of deletion mutants were produced, such as NS3delN1–20, NS3delN1–40, NS3delN1–70, NS3delN1–70, and NS3HadN40–70 (Fig. 4). RdRp reactions containing NS5B and either of those deletion mutants were performed. Quantification of synthesized RNA was determined by using real-time RT-PCR as described in Fig. 1. Results showed that NS3delN1–20, NS3delN1–40 and NS3HadN40–70 still stimulated NS5B RdRp activity, and the stimulations were in agreement with increasing amount of those purified deletion mutants,
Fig. 1. Effects of full-length and truncated NS3 proteins on RdRp activity of NS5B. RdRp assays containing NS5B (100 ng) and increasing amounts of NS3F(A), NS3H (B) or NS3P (C) (0, 100, 150, 200, 250,300, and 350 ng, respectively) were performed as described previously (Xiao et al., 2004). The CSFV 3 UTR (228 nt) served as a template. Quantification of synthesized RNA was determined by using real-time RT-PCR. To normalize data, the amount of CSFV RNA in each sample was divided by the amount detected in the NS5B RdRp assay in the absence of NS3 and its truncated forms under same conditions.
while NS3delN1–70 and NS3delN40–70 did not increase NS5B RdRp activity (Fig. 5 and results not shown). These results indicated that stimulations of NS5B RdRp activity by NS3 still was efficient when 20 and 40 amino acids were deleted from the N-terminal end of NS3 protease domain, respectively and implied that the 31 amino acids were from positions 40 to 70 at the N-terminal end of NS3 protease domain were important to stimulation of NS5B RdRp activity by NS3. 3.4. NS3–NS5B interaction by a 31-amino acid fragment in NS3 protease domain Furthermore, the GST-pull-down assay and the coimmunoprecipitation analysis containing NS5B and either of the
20
P. Wang et al. / Virus Research 148 (2010) 17–23
Fig. 2. GST-pull-down analysis of NS3–NS5B interactions NS3F, NS3H or NS3P was mixed with GST-NS5B, or GST (as a control), immobilized on GST resin. Each bound protein was determined by Western blot analysis with anti-NS3 monoclonal antibody. Purified NS3F, NS3H and NS3P were used as positive controls. The sizes of protein molecular mass markers are indicated on the left.
above deletion NS3 mutants was carried out. GST-pull-down assay results show that NS3delN1–20, NS3delN1–40 and NS3HadN40–70 were pulled down with NS5B (Fig. 6, lanes 3, 4 and 7), respectively, as NS3F (lane 2), but NS3delN1–70 and NS3delN40–70 were not (lanes 5 and 6), suggesting that the 31-amino acid fragment at the N-terminal end (positions 40–70) of NS3 protease domain mediated the specific NS3–NS5B interaction. Indeed, when PK-15
Fig. 4. Schematic representation of NS3 protein and its different deleted forms. Positions of amino acids (1590, 1764 and 2272) are shown over NS3 protein. NS3H and NS3P are shown down NS3 protein. The internal deletions were indicated by “V”-shape line. NS3delN1–20, NS3delN1–40, and NS3delN1–70 are the truncated NS3 proteins with deletion of 20, 40 and 70 amino acids from the N-terminal end of full-length NS3 protein, respectively. NS3delN40–70 is the truncated NS3 protein with deletion of 31 amino acids from positions 40–70 at the N-terminal end. NS3HadN40–70 is the lengthened NS3H with addition of 31 amino acids from positions 41–70 to the N-terminal end. A 31-amino acid fragment from positions 40 to 70 was shown below, and the amino acid “His (H)” was indicated by an arrow.
cells were transiently cotransfected with the pcDNA3.1 vector containing NS5B and either of the pcDNA3.1/N-FLAG vectors containing those NS3 deletion mutants, respectively, CSFV NS5B was observed to interact with NS3delN1–20, NS3delN1–40 and NS3HadN40–70 in PK-15 cells (Fig. 7, lanes 4, 6, 12), in which, CSFV NS5B did not interacted with NS3delN1–70 and NS3delN40–70 (lanes 8 and 10). Those data indicated that CSFV NS3 was bound to NS5B by the 31-amino acid fragment located in the NS3 protease domain.
Fig. 3. Co-immunoprecipitation analysis of NS3–NS5B interactions PK-15 cells were cotransfected with the pcDNA3.1 vector containing NS5B and either of the pcDNA3.1/NFLAG vector containing NS3F (lane 2), NS3H (lane 4), and NS3P (lane 6). As a control, PK-15 cells were also transfected only with the pcDNA3.1/N-FLAG vector containing NS3F (lane 1), NS3H (lane 3), or NS3P (lane 5). (A) Total lysates were fractionated by SDS-PAGE (10%) and subjected to Western blot analysis with anti-FLAG. (B) In vivo co-immunoprecipitated of NSF, NS3H and NS3P with NS5B was probed with anti-FLAG antibody (upper panel). Efficient immunoprecipitation of NS5B was also shown (lower panel).
P. Wang et al. / Virus Research 148 (2010) 17–23
21
Fig. 6. GST-pull-down analysis of different deleted NS3–NS5B interactions NS3delN1–20, NS3delN1–40, NS3delN1–70, NS3delN40–70 or NS3HadN40–70 was mixed with GST-NS5B, or GST (as a control), immobilized on GST resin. Each bound protein was determined by Western blot analysis with anti-NS3 monoclonal antibody. Purified NS3delN1–20, NS3delN1–40, NS3delN1–70, NS3delN40–70 and NS3HadN40–70 were used as positive controls. The sizes of protein molecular mass markers are indicated on the left.
Fig. 5. Effects of different deleted NS3 proteins on RdRp activity of NS5B RdRp assays containing NS5B (100 ng) and increasing amounts of NS3delN1–20 (A), NS3delN1–40 (B), or NS3HadN40–70 (C) (0, 100, 150, 200, 250,300, and 350 ng, respectively) were performed as described in Fig. 1.
4. Discussion For HCV, there is a controversy on the role of NS3 in enhancing NS5B RdRp activity. One observed that the HCV NS3 protein enhances NS5B RdRp activity (Piccininni et al., 2002). In contrast, another found that the full-length HCV NS3 does not increase NS5B RdRp activity (Zhang et al., 2005). To investigate the effect of NS3 on CSFV NS5B RdRp activity, RdRp reactions containing NS5B and NS3 proteins were performed. The CSFV 3 UTR was served as a template so that experiment results can reveal actual situation more authentically. We found that the full-length NS3 stimulated NS5B RdRp activity in a dose-dependent manner, but the truncated NS3 protein (NS3H) with helicase domain and another truncated NS3 protein (NS3P) with NS3 protease domain did not (Fig. 1). About the role of CSFV NS3 in RNA synthesis, our results are consistent
Fig. 7. Co-immunoprecipitation analysis of deletion NS3 mutant-NS5B interactions PK-15 cells were cotransfected with the pcDNA3.1 vector containing NS5B and either of the pcDNA3.1/N-FLAG vector containing NS3F (lane 2), NS3delN1–20 (lane 4), NS3delN1–40 (lane 6), NS3delN1–70 (lane 8), NS3delN40–70 (lane 10) or NS3HadN40–70 (lane 12). As a control, PK-15 cells were also transfected only with the pcDNA3.1/N-FLAG vector containing NS3F (lane 1), NS3delN1–20 (lane 3), NS3delN1–40 (lane 5), NS3delN1–70 (lane 7), NS3delN40–70 (lane 9) or NS3HadN40–70 (lane 11). The sizes of protein molecular mass markers are indicated on the left. Total lysates were fractionated by SDS-PAGE (10%) and subjected to Western blot analysis with anti-FLAG (upper panel). In vivo co-immunoprecipitated of NS3F, NS3delN1–20, NS3delN1–40, NS3delN1–70, NS3delN40–70, and NS3HadN40–70 with NS5B was probed with anti-FLAG antibody (middle panel). Efficient immunoprecipitation of NS5B was also shown (lower panel).
22
P. Wang et al. / Virus Research 148 (2010) 17–23
with those reported previously for HCV (Piccininni et al., 2002). The immunoprecipitation results, together with those of the GSTpull-down assays, show that CSFV NS5B is able to interact with either of the full-length NS3 and the NS3 protease domain, and not interact with NS3H (Figs. 2 and 3), implying that stimulation of NS5B RdRp activity is obtained by CSFV NS3 binding to the NS5B through the NS3 protease domain. It has been reported that the HCV NS3 protease domain is required for specific HCV NS3 and NS5B interaction (Zhang et al., 2005), and HCV NS5B forms a complex with NS3 through an amino-terminal portion of NS3 (Ishido et al., 1998). The interaction might up-regulate HCV NS3 helicase activity (Wen et al., 2009; Zhang et al., 2005). Our previous reports suggested that CSFV NS5B significantly enhanced the stimulative effect of NS3 on both IRES-mediated and cellular translation via the protease domain (Xiao et al., 2008). Our present data show that CSFV NS3 forms a complex with NS5B through NS3 protease domain and stimulates NS5B activity. Recent evidence has indicated that CSFV NS5B, in combination with NS3, functions in viral replication. It has been reported that rescue of virus and recover of viral RNA synthesis are not observed in the complementation experiments including either CSFV NS5B mutant or NS3 mutant (Sheng et al., 2010), suggesting that the functions of these proteins of CSFV might be cis. Both CSFV NS5B and NS3 proteins have been shown to preferentially bind to the authentic viral 3 UTR (Xiao et al., 2004; Sheng et al., 2007). Considering the fact that BVDV NS5B and NS3 have been demonstrated to be membrane-associated, with a key role for formation of the replication-transcription complex (Lai et al., 1999; Zhang et al., 2003), both CSFV NS5B and NS3 might also be membrane-associated. The fact that neither NS3H nor NS3P alone stimulate NS5B RdRp activity indicated that the full-length NS3 is required for stimulation of NS5B RdRp activity. To stimulate NS5B RdRp activity, NS3P was served as a NS5B-binding site. Furthermore, to map important regions of NS3 protease domain for stimulation of NS5B RdRp activity, a series of deletion were introduced into the NS3 protease domain (Fig. 4). The data from RdRp reactions, GST-pull-down assays and coimmunoprecipitation analysis indicated that a 31-amino acid fragment from positions 40 to 70 at the terminal end of NS3 protease domain is important for stimulation of NS5B RdRp activity and NS3–NS5B interaction (Figs. 5–7). Indeed, the truncated NS3 mutant (NS3HadN40–70) only consisting of NS3H and the 31-amino acid fragment still enhances NS5B RdRp activity (Figs. 4 and 5). Moreover, the NS5B-binding ability of the NS3HadN40–70 observed in the GST-pull assays and coimmunoprecipitation analyses is well correlated with its positive effect on NS5B RdRp activity (Figs. 5–7), indicating that CSFV NS3 stimulates NS5B RdRp activity by binding directly to NS5B through the 31-amino acid fragment located in the NS3 protease domain. We analyzed the amino acid composition of the fragment. It was found that there were 20 polar and hydrophilic ones among 31 amino acids, which is one of the properties of protein binding sites because protein–protein interfaces are often studded with many polar residues or hydrophilic residues (Hu et al., 2000; Xu et al., 1997b,c). Moreover, at the interface, there are higher proportions of buried charged and polar residues as compared to protein cores, suggesting that hydrogen bonds and ion pairs contribute to the stability of protein binding (Keskin et al., 2008). Furthermore, by an alignment analysis we found that the conserved amino acid “His (H)” at position 39 was located in the 31-amino acid fragment (Fig. 4 and results not shown). It is one of those residues predicted to form the classical serine protease catalytic triad (Ryan et al., 1998), which might also be important for a protein binding site. Although our results about the role of CSFV NS3 in RNA synthesis are consistent with those reported for HCV in the paper of Piccininni et al. (2002), our present conclusion that CSFV NS3 RNA helicase does not increase NS5B RdRp activity is contrary to that
from the paper, in which, HCV NS3 helicase domain has been found to alone enhance RNA synthesis. We speculate that the difference is due to virus species, but without experimental evidence support the proposal. Although HCV NS3 RNA helicase is necessary for viral replication (Lam and Frick, 2006), the NS3 protease domain is also important to RNA helicase. It has been found in HCV that NS3 protease domain is required for RNA unwinding by NS3, and when the NS3 protein lacks the protease domain it unwinds RNA more slowly (Beran et al., 2007; Frick et al., 2004). Therefore, a fulllength NS3 protein might be important not only for NS5B RdRp activity and also for NS3 RNA helicase in HCV and CSFV. The role of NS3 in the cytopathogenicity and life cycle of CSFV is also important. The cytopathogenicity of pestiviruses including CSFV has been demonstrated to correlate with the expression of large amounts of NS3 (Aoki et al., 2004; Gallei et al., 2008; Meyers and Thiel, 1995). Therefore, NS3 is regarded as marker protein for cytopathogenic pestiviruses. The data from complementation experiments have showed that NS2–3 and NS3, each in association with NS4A, have independent functions in the CSFV life cycle (Moulin et al., 2007). An essential function for NS3 in pestiviral RNA replication which cannot be supplied by its NS2–3 precursor has been implied in BVDV replication analysis (Lackner et al., 2004). Taken together, we found that CSFV NS3 protease is the place to mediate a NS3–NS5B interaction for stimulation of NS5B RdRp activity. The 31-amino acid fragment localized at positions 40–70 of the NS3 protease might be the NS5B-binding site. This is important for understanding the mechanism for CSFV replication. However, which region of NS5B is the NS3-binding site remains to be elucidated. Acknowledgements This work was supported by the National Natural Science Foundation of China (30670445, 30870492), the Shanghai Municipal Science and Technology Commission (07DZ12038, 08JC1416900), the Shanghai Municipal Education Commission (08ZZ66), the Shanghai Leading Academic Discipline Project (S30406) and the Leading Academic Discipline Project of Shanghai Normal University (DZL709). References Aoki, H., Sakoda, Y., Nakamura, S., Suzuki, S., Fukusho, A., 2004. Cytopathogenicity of classical swine fever viruses that do not show the exaltation of Newcastle disease virus is associated with accumulation of NS3 in serumfree cultured cell lines. J. Vet. Med. Sci. 66, 161–167. Becher, P., Thiel, H.-J., 2002. Genus Pestivirus (Flaviviridae). In: Tidona, C.A., Darai, G. (Eds.), The Springer Index of Viruses. Springer-Verlag, Heidelberg, Germany, pp. 327–331. Beran, R.K., Serebrov, V., Pyle, A.M., 2007. The serine protease domain of hepatitis C viral NS3 activates RNA helicase activity by promoting the binding of RNA substrate. J. Biol. Chem. 282, 34913–34920. Cuthbert, J.A., 1994. Hepatitis C: progress and problems. Clin. Microbiol. Rev. 7, 505–532. Dimitrova, M., Imbert, I., Kieny, M.P., Schuster, C., 2003. Protein–protein interactions between hepatitis C virus nonstructural proteins. J. Virol. 77, 5401–5414. Frick, D.N., Rypma, R.S., Lam, A.M., Gu, B., 2004. The nonstructural protein 3 protease/helicase requires an intact protease domain to unwind duplex RNA efficiently. J. Biol. Chem. 279, 1269–1280. Gallei, A., Blome, S., Gilgenbach, S., Tautz, N., Moennig, V., Becher, P., 2008. Cytopathogenicity of classical swine fever virus correlates with attenuation in the natural host. J. Virol. 82, 9717–9729. Gong, Y., Trowbridge, R., Macnaughton, T.B., Westaway, E.G., Shannon, A.D., Gowans, E.J., 1996. Characterization of RNA synthesis during a one-step growth curve and of the replication mechanism of bovine viral diarrhea virus. J. Gen. Virol. 77, 2729–2736. Gu, B., Liu, C., Lin-Goerke, J., Maley, D.R., Gutshall, L.L., Feltenberger, C.A., Del Vecchio, A.M., 2000. The RNA helicase and nucleotide triphosphatase activities of the bovine viral diarrhea virus NS3 protein are essential for viral replication. J. Virol. 74, 1794–1800. Heinz, F.X., Collett, M.S., Purcell, R.H., Gould, E.A., Howard, C.R., Houghton, M., Moormann, R.J.M., Rice, C.M., Thiel, H.-J., 2000. Family Flaviviridae. In: Fauquet, C.M., van Regenmortel, M.H.V., Bishop, D.H.L., Carstens, E.B., Estes, M.K., Lemon, S.M.,
P. Wang et al. / Virus Research 148 (2010) 17–23 Maniloff, J., Mayo, M.A., McGeoch, D.J., Pringle, C.R., Wickner, R.B. (Eds.), Virus Taxonomy: Seventh Report of the International Committee on Taxonomy of Viruses. Academic Press, Inc., San Diego, CA, pp. 859–878. Hu, Z., Ma, Buyong, Wolfson, H., Nussinov, R., 2000. Conservation of polar residues as hot spots at protein interfaces. Proteins: Struct. Funct. Bioinform. 39, 331–342. Ishido, S., Fujita, T., Hotta, H., 1998. Complex formation of NS5B with NS3 and NS4A proteins of hepatitis C virus. Biochem. Biophys. Res. Commun. 244, 35–40. Lam, A.M., Frick, D.N., 2006. Hepatitis C virus subgenomic replicon requires an active NS3 RNA helicase. J. Virol. 80, 404–411. Keskin, O., Gursoy, A., Ma, B., Nussinov, R., 2008. Principles of protein–protein interactions: what are the preferred ways for proteins to interact? Chem. Rev. 108, 1225–1244. Kolykhalov, A.A., Mihalik, K., Feinstone, S.M., Rice, C.M., 2000. Hepatitis C virus-encoded enzymatic activities and conversed RNA elements in the 3 nontranslated region are essential for virus replication in vivo. J. Virol. 74, 2046–2051. Lackner, T., Müller, A., Pankraz, A., Becher, P., Thiel, H.J., Gorbalenya, A.E., Tautz, N., 2004. Temporal modulation of an autoprotease is crucial for replication and pathogenicity of an RNA virus. J. Virol. 78, 10765–10775. Lai, V.C.H., Kao, C.C., Ferari, E., Park, J., Uss, A.S., Wright-Minogue, J., Hong, Z., Lai, J.Y.N., 1999. Mutational analysis of bovine viral diarrhea virus RNA-dependent RNA polymerase. J. Virol. 73, 10129–10136. Lindströma, H., Lundina, M., Häggströma, S., Persson, M.A.A., 2006. Mutations of the hepatitis C virus protein NS4B on either side of the ER membrane affect the efficiency of subgenomic replicons. Virus Res. 121, 169–178. Lundin, M., Monne, M., Widell, A., von Heijne, G., Persson, M.A.A., 2003. Topology of the membrane-associated hepatitis C virus protein NS4B. J. Virol. 77, 5428–5438. Masaki, T., Suzuki, R., Murakami, K., Aizaki, H., Ishii, K., Murayama, A., Date, T., Matsuura, Y., Miyamura, T., Wakita, T., 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. Meyers, G., Thiel, H.J., 1995. Cytopathogenicity of classical swine fever virus caused by defective interfering particles. J. Virol. 69, 3683–3689. Moennig, V., Plagemann, P.G.W., 1992. The pestiviruses. Adv. Virus Res. 41, 53–98. Moulin, H.R., Seuberlich, T., Bauhofer, O., Bennett, L.C., Tratschin, J.D., Hofmann, M.A., Ruggli, N., 2007. Nonstructural proteins NS2–3 and NS4A of classical swine fever virus: essential features for infectious particle formation. Virology 365, 376– 389. Piccininni, S., Varaklioti, A., Nardelli, M., Dave, B., Raney, K.D., McCarthy, J.E., 2002. Modulation of the hepatitis C virus RNA-dependent RNA polymerase activity by the non-structural (NS) 3 helicase and the NS4B membrane protein. J. Biol. Chem. 277, 45670–45679. Ryan, M.D., Monaghan, S., Flint, M., 1998. Virus-encoded proteinases of the Flaviviridae. J. Gen. Virol. 79, 947–959. Sheng, C., Xiao, M., Geng, X., Liu, J., Wang, Y., Gu, F., 2007. Characterization of interaction of classical swine fever virus NS3 helicase with 3 untranslated region. Virus Res. 129, 43–53. Sheng, C., Zhua, Z., Yua, J., Wan, L., Wang, Y., Chen, J., Gu, F., Xiao, M., 2010. Characterization of NS3, NS5A and NS5B of classical swine fever virus through mutation and complementation analysis. Vet. Microbiol. 140, 72–80.
23
Steffen, S., Thiel, H.J., Behrens, S.E., 1999. The RNA-dependent RNA polymerase of different members of the family Flaviviridae exhibits similar properties in vitro. J. Gen. Virol. 80, 2583–2590. Suzich, J.A., Tamura, J.K., Palmer-Hill, F., Warrener, P., Grakoui, A., Rice, C.M., Feinstone, S.M., Collett, M.S., 1993. Hepatitis C virus NS3 protein polynucleotidestimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes. J. Virol. 67, 6152–6158. Tellinghuisen, T.L., Marcotrigiano, J., Gorbalenya, A.E., Rice, C.M., 2004. The NS5A protein of hepatitis C virus is a zinc metalloprotein. J. Biol. Chem. 279, 48576–48587. Tellinghuisen, T.L., Paulson, M.S., Rice, C.M., 2006. The NS5A protein of bovine viral diarrhea virus contains an essential zinc-binding site similar to that of the hepatitis C virus NS5A protein. J. Virol. 80, 7450–7458. Wang, Y., Xiao, M., Chen, Jun., Zhang, W., Luo, J., Bao, K., Nie, M., Chen, J., Li, Bo., 2007. Mutational analysis of the GDD sequence motif of classical swine fever virus RNA-dependent RNA polymerases. Virus Genes 34, 63–65. Warrener, P., Collett, M.S., 1995. Pestivirus NS3 (p80) protein possesses helicase activity. J. Virol. 69, 1720–1726. Wen, G., Xue, J., Shen, Y., Zhang, C., Pan, Z., 2009. Characterization of classical swine fever virus (CSFV) nonstructural protein 3 (NS3) helicase activity and its modulation by CSFV RNA-dependent RNA polymerase. Virus Res. 141, 63–70. Xiao, M., Chen, J., Li, B., 2003. RNA-dependent RNA polymerase activity of classical swine fever virus NS5B protein expressed in natural host cells. Acta Virol. 47, 17–26. Xiao, M., Zhang, C.Y., Pan, Z.S., Wu, H.X., Guo, J.Q., 2002. Classical swine fever virus NS5B-GFP fusion possesses an RNA-dependent RNA polymerase activity. Arch. Virol. 147, 1779–1785. Xiao, M., Gao, J., Wang, W., Wang, Y., Chen, J., Chen, J., Li, B., 2004. Specific interaction between the classical swine fever virus NS5B protein and the viral genome. Eur. J. Biochem. 271, 3888–3896. Xiao, M., Li, H., Wang, Y., Wang, X., Wang, W., Peng, J., Chen, J., Li, B., 2006. Characterization of the N-terminal domain of classical swine fever virus RNA-dependent RNA polymeras. J. Gen. Virol. 87, 347–356. Xiao, M., Bai, Y., Xu, H., Geng, X., Chen, J., Wang, Y., Chen, J., Li, B., 2008. Effect of NS3 and NS5B protein on classical swine Fever Virus IRES-mediated translation and its host cellular translation. J. Gen. Virol. 89, 994–999. Xu, J., Mendez, E., Caron, P.R., Lin, C., Murcko, M.A., Collett, M.C., Rice, C.M., 1997a. Bovine viral diarrhea virus: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication. J. Virol. 71, 5312–5322. Xu, D., Lin, S.L., Nussinov, R., 1997b. Protein–protein interfaces are studded with many polar residues or hydrophilic residues. J. Mol. Biol. 265, 68–84. Xu, D., Tsai, C.J., Nussinov, R., 1997c. Hydrogen bonds and salt bridges across protein–protein interfaces. Protein Eng. 10, 999–1012. Zhang, C., Kim, Y.C., Kumar, R., Yuan, F., Shi, P.Y., Kao, C., Luo, G., 2005. Stimulation of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase activity by the NS3 protease domain and by HCV RNA-dependent RNA polymerase. J. Virol. 79, 8687–8697. Zhang, G., Flick-Smith, H., McCauley, J.W., 2003. Differences in membrane association and sub-cellular distribution between NS2–3 and NS3 of bovine viral diarrhoea virus. Virus Res. 97, 89–102.
Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Classical swine fever virus NS3 enhances RNA-dependent RNA polymerase activity by binding to NS5B Ping Wang, Yujing Wang, Yu Zhao, Zailing Zhu, Jialin Yu, Lingzhu Wan, Jun Chen, Ming Xiao ∗ Research Institute for Agricultural Microbiology, College of Life and Environment Sciences, Shanghai Normal University, Shanghai, 200234, China
a r t i c l e
i n f o
Article history: Received 12 September 2009 Received in revised form 24 November 2009 Accepted 25 November 2009 Available online 29 November 2009 Keywords: Classical swine fever virus NS3 NS5B NS3–NS5B interaction
a b s t r a c t NS3 of pestiviruses contains a protease domain and a RNA helicase/NTPase domain. Contradictory results have been reported regarding NS3 in RNA synthesis. To investigate the effect of NS3 on classical swine fever virus (CSFV) NS5B RNA-dependent RNA polymerase activity (RdRp) activity and NS3–NS5B interaction, RdRp reactions, GST-pull-down assays and co-immunoprecipitation analyses containing NS5B and either of NS3 protein and the different truncated NS3 mutants were performed, respectively. We found that NS3 stimulated NS5B RdRp activity in a dose-dependent manner by binding to NS5 through a NS3 protease domain. Furthermore, mapping important regions of the NS3 protease domain was carried out by deletion mutagenesis, associated with RdRp reactions, GST-pull-down assays and coimmunoprecipitation analyses. Results showed that stimulation of CSFV NS5B RdRp activity was obtained by NS3 binding to NS5B through a 31-amino acid fragment at the N-terminal end of NS3 protease domain, which mediated a specific NS3–NS5B interaction. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Classical swine fever virus (CSFV) is a small enveloped virus, which is an important pathogen of pigs, and often causes major losses in stock farming. CSFV is a member of the genus Pestivirus which also comprises bovine viral diarrhea virus 1 (BVDV-1), BVDV2, and border disease virus (BDV) (Becher and Thiel, 2002; Heinz et al., 2000). The genus Pestivirus belongs to the family Flaviviridae. The hepatitis C virus (HCV) also belongs to this family (Cuthbert, 1994). CSFV genome is a single plus-strand RNA, and contains a single large open reading frame (ORF), a 5 untranslated region (5 UTR) and a 3 untranslated region (3 UTR). The ORF encodes a polyprotein of approximately 3900 amino acids. The viral genome enters host cell and here is translated into the polyprotein, which is processed by viral and cellular proteases into 12 mature proteins (4 structural proteins and 8 nonstructural proteins): Npro , P70, NS2, NS3, NS4A, NS4B, NS5A, NS5B (Moennig and Plagemann, 1992). The CSFV NS5B protein has an RNA-dependent RNA polymerase (RdRp) activity (Steffen et al., 1999; Xiao et al., 2002, 2006), contains a conserved GDD motif necessary for the catalytic activity (Wang et al., 2007), transcribes the genome into minus strands, which serve as templates to produce more plusstrand RNAs for packaging into progeny viral capsids (Gong et al., 1996). During RNA synthesis, replicative intermediate and replica-
∗ Corresponding author. Tel.: +86 21 64321022; fax: +86 21 65642468. E-mail address: [email protected] (M. Xiao). 0168-1702/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2009.11.015
tive forms have been detected (Gong et al., 1996). Recent evidence has indicated that NS3 protein is also important in viral replication (Gu et al., 2000; Kolykhalov et al., 2000; Piccininni et al., 2002; Sheng et al., 2007). NS3 is a multifunctional protein possessing serine protease, RNA helicase, and nucleoside triphosphatase (NTPase) activities located in two functionally distinct domains. The N-terminal one-third of pestiviral NS3 primarily serves as a protease to process the viral polyprotein (Xu et al., 1997a). The helicase and NTPase activities are localized to the C-terminal of NS3 protein (Suzich et al., 1993; Warrener and Collett, 1995). It has been reported that NS5A protein is an essential component of the viral RNA replication machinery and may also function in modulation of the host cell environment (Masaki et al., 2008; Tellinghuisen et al., 2004, 2006). In addition, NS4B and NS4A are vital for viral replication (Lindströma et al., 2006; Lundin et al., 2003; Moulin et al., 2007). However, interaction of these proteins and the effects of those interactions on viral replication remain poorly understood. In this paper, we investigated the NS3–NS5B interaction and the effect of this interaction on CSFV RdRp activity by deletion mutagenesis of NS3 protein. 2. Materials and methods 2.1. Protein expression and purification Prokaryotic expression of CSFV NS5B protein, NS3 and its truncated forms were performed as described previously (Sheng et al., 2007; Xiao et al., 2006). The cDNAs encoding full-length NS3 (NS3F) (amino acids 1590–2272), truncated NS3 protease
18
P. Wang et al. / Virus Research 148 (2010) 17–23
(NS3P) (amino acids 1590–1763), truncated NS3 helicase (NS3H) (amino acids 1764–2272), NS3delN1–20 (amino acids 1610–2272), NS3delN1–40, (amino acids 1630–2272), NS3delN1–70 (amino acids 1660–2272), and NS5B (amino acids 3181–3898) were obtained by RT-PCR from the genome of CSFV Shimen strain, and cloned into pET28(a) vector, respectively. Expression of NS3delN40–70 and NS3HadN40–70 were obtained by deletion of amino acids 40–70 and by deletion of amino acids 1–39 and 71–174 from N-terminal end of NS3 (Fig. 4). A His6 tag was added to the C terminus of each of these proteins to facilitate protein. The inserted regions of all clones were sequenced through dideoxynucleotide sequencing and no changes were found. These resulting plasmids were introduced into the Escherichia coli strain BL21(DE3) for expression driven by T7 RNA polymerase. Expression was induced by addition of isopropylthiogalactoside (IPTG). The bacterial cell culture was harvested by centrifugation at 6000 × g for 10 min, and washed in phosphate-buffered saline (PBS). The cells from 1000 ml were resuspended in 20 ml of the buffer containing 50 mM Na-phosphate [pH 8.0], 300 mM NaCl, 10 mM imidazole, 10 mM -mercaptoethanol, 10% glycerol, 1% Nonidet P-40, supplemented with 1 mM phenylmethylsulfonyl fluoride and 10 mM leupeptin. Cells were lysed by freezing and thawing, followed by sonication. The cleared lysates were obtained by centrifugation at 35,000 × g for 15 min, and then, purified using nickel–nitrilotriacetic acid (Ni–NTA)–sepharose resin (Gibco BRL) following the manufacturer’s direction. These CSFV proteins were collected and dialyzed in the buffer A (50 mM Tris–HCl [pH 8.0], 1 mM DTT, 50 mM NaCl, 5 mM MgCl2 , 10% glycerol), respectively. These protein solutions and dilutions of bovine serum albumin with known concentration were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%). The gels with the samples were stained with Coomassie brilliant blue. The amount of each of these proteins was determined by densitometry scanning and comparing the two samples on the same gel. Purified proteins were separated by SDS-PAGE. To express a GST-NS5B fusion protein, the fragments encoding NS5B (amino acids 3181–3898) were amplified by PCR, engineered with a BamHI site and an XhoI site. PCR products were digested with BamHI/XhoI and inserted into a pGEX-4T-1 vector (Amersham Pharmacia Biotech), in frame with the GST-encoding sequence, to generate pGEX-4T/NS5B. The GST or GST-NS5B fusion protein was expressed in Escherichia coli BL21(DE3), purified with glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) according to the manufacturer’s instructions, as described previously (Dimitrova et al., 2003). In brief, expression was induced by addition of IPTG. The cells from 500 ml of culture were then harvested and sonicated in 45 ml of PBS buffer supplemented with a protease inhibitor cocktail and 1% Triton X-100. Insoluble materials were pelleted at 15,000 × g for 10 min, and 500 l of a 50% slurry of glutathione-Sepharose 4B beads was added to the clarified supernatants. The beads were allowed to bind proteins for 1 h, washed three times in PBS, and finally resuspended in 500 l of PBS supplemented with 1% Triton X-100. 2.2. RdRp assays RdRp assays were performed essentially as described previously (Xiao et al., 2004). Total volume was 50 ml, containing the following supplements: 50 mM Hepes (pH 8.0), 5 mM MgCl2 , 10 M DTT, 25 mM KCl, l mM EDTA, 20U RNasin, 50 g actinomycin D (Sigma), 200 M each NTP, 1 l of RNA template (250 ng/ml), 100 ng NS5B proteins and increasing amounts of NS3F, NS3H or NS3P (0, 100, 150, 200, 250,300, and 350 ng, respectively). The mixture was incubated at 37 ◦ C for 1 h, and the reaction was stopped by the addition of 2 l of EDTA (200 mM). The reaction samples were extracted with phenol/chloroform, and RNAs were precipitated with isopropyl alcohol.
2.3. Quantification of viral RNA Quantification of viral RNA was determined by real-time RT-PCR. Forward primer (nt 12071–12093, 5 GCGCGGGTAACCCGGGATCTGAA-3 ), reverse primer (nt 12174–12190, 5 -CAGTTCTTACTCATTCA-3 ), and TaqMan probe (nt 12103–12124, FAM-5 -AGGACCCTATTGTAGATAACAC-3 -TAMRA) were designed based on the 3 UTR sequence of CSFV Shimen strain (accession number: AF092448). Total RNA was extracted from products of RdRp assays with phenol/chloroform. After ethanol precipitation, the RNA was dried, and redissolved in 20 l of double distilled H2 O. The concentration of RNA was determined by measuring its absorbance at 260 nm. RT was performed using Superscript II reverse transcriptase (Invitrogen). The quantitative real-time PCR was run using the AbiPrism7000 sequence detection system (Applied Biosystems). PCR were performed for 40 cycles with cycling conditions of 15 s at 95 ◦ C and 1 min at 60 ◦ C. In vitro-transcribed CSFV RNA of known concentrations were run in parallel reactions, used to generate a standard curve. 2.4. Cell culture PK-15 cells, the natural host of CSFV were used for expressing the CSFV NS5B protein. They were obtained from the China Center for Type Culture Collection, Wuhan. The cells were cultured in DMEM (Gibco BRL) supplemented with 10% of fetal calf serum, penicillin (100 U/ml) and streptomycin (100 g/ml) at 37 ◦ C in a humidified 5% CO2 atmosphere. 2.5. GST-pull-down assay Approximately 2 g of GST or GST-NS5B fusion protein immobilized on 20 l of GST resin was preblocked with 1% bovine serum albumin and then incubated with 2 g of NS3 wild type or mutants in 300 l of PBST buffer (phosphate-buffered saline containing 1% Triton X-100) for 3 h on a rotating device at room temperature. After washing with PBST, the bound proteins were fractionated by SDSPAGE (10%) and subjected to Western blot analysis with anti-NS3 monoclonal antibody. 2.6. Co-immunoprecipitation analysis For co-immunoprecipitation analyses, the sequences encoding NS3F, NS3H, NS3P, NS3delN1–20, NS3delN1–40, NS3delN1–70, NS3delN40–70, and NS3HadN40–70 were obtained by PCR from the pET28(a) vectors containing these protein-encoding regions used for above prokaryotic expression, engineered with BamHI and XbaI sites and cloned into a pcDNA3.1/N-FLAG vector. CSFV NS5B (amino acids 3181–3898) was cloned into pcDNA3.1 vector as described previously (Xiao et al., 2003). The resulting recombinant vectors were used for co-transfection of PK-15 cells, respectively. PK-15 cells transfected with the above plasmids were harvested, washed and sonicated in RIPA buffer consisting of 150 mM NaCl, 0.5% Triton X-100, 10 mM Triton-HCl (pH 7.5). Lysates were centrifuged at 12,000 × g for 10 min at 4 ◦ C and the supernatants was immunoprecipitated with appropriate antibodies for 1 h at 4 ◦ C, followed by incubation with 20 l protein G-agarose beads (Pharmacia) at 4 ◦ C for 1 h. After washing with RIPA buffer, the bound proteins were eluted, fractionated by SDS-PAGE (10%), transferred onto nitrocellulose membranes, and subjected to Western blot analysis with anti-FLAG monoclonal antibody or anti-NS5B antibody. Protein bands were visualized by enhanced chemiluminescence according to the manufacturer’s instructions (Amersham Pharmacia Biotech).
P. Wang et al. / Virus Research 148 (2010) 17–23
19
3. Results 3.1. Stimulation of NS5B RdRp activity by NS3 To examine the effects of NS3 on CSFV NS5B polymerase activity, we expressed the viral NS5B protein, full-length NS3 protein (NS3F), the truncated NS3 protein with postulated helicase domain (NS3H) and another truncated NS3 protein (NS3H) with NS3 protease (NS3P). The NS5B RdRp activities were tested in the presence of increasing amount of NS3F, NS3H and NS3P, respectively. The 3 UTR of CSFV Shimen strain genome was served as a template. To exclude the possibility that the terminal nucleotidyl transferase activity of CSFV NS5B influences the value for its RdRp activity, we used a 3 UTR with oxidized 3 -terminal hydroxyl group (Xiao et al., 2004, 2006). Quantification of synthesized RNA was determined by using real-time RT-PCR. To normalize data, the amount of CSFV RNA in each sample was divided by the amount detected in the NS5B RdRp assay in the absence of NS3 and its truncated forms under same conditions. The experiment was repeated three times. As shown in Fig. 1, NS3F stimulated RdRp activity, and the stimulation was in agreement with increasing amount of purified NS3F, while NS3H and NS3P did not increase NS5B RdRp activity. These results suggested that stimulation of NS5B RdRp activity by NS3 protein needed NS3 helicase domain and NS3 protease domain, and there might be an interaction between NS5B and NS3, NS3 helicase or NS3 protease domain. 3.2. NS3–NS5B interaction by NS3 protease domain To elucidate whether NS5B interacts with NS3 or its truncated forms, a GST-pull-down assay was performed. NS3F, NS3H or NS3P was mixed with the CSFV NS5B bound to glutathione-agarose, and GST protein alone was served as a control. Pulled down proteins were fractioned by SDS-PAGE and subjected to Western blot analysis. Results showed that NS3F was pulled down with GST-NS5B, not with GST alone (Fig. 2, lanes 2 and 1), indicating a specific NS3–NS5B interaction. It was found that NS3P was pulled down with GST-NS5B, but NS3H was not (lanes 4 and 3), implying that the NS3 protease domain mediated the NS3–NS5B interaction, and the NS3 helicase domain did not. To determine whether the interaction of NS5B and either of NS3 and its truncated forms occurs in the cell, co-immunoprecipitation analyses were performed. PK15 cells were transiently cotransfected with the pcDNA3.1 vector containing NS5B and either of the pcDNA3.1/N-FLAG vectors containing NS3 and its truncated forms. Coprecipitated proteins were detected by Western blot analysis. A specific interaction between the NS5B and either of NS3F and NS3P was observed, indicating that CSFV NS5B interacted with either of the full-length NS3 and the NS3 protease domain in PK-15 cells (Fig. 3). These results strongly suggested that stimulation of CSFV NS5B RdRp activity was obtained by NS3 binding to NS5B, and the NS5B-binding site on NS3 might be located in the NS3 protease domain. 3.3. Influence of deletion NS3 mutants on NS5B RdRp activity To determine which region of NS3 protease domain is important to stimulation of NS5B RdRp activity, deletion mutagenesis of NS3 protease domain was carried out. A series of deletion mutants were produced, such as NS3delN1–20, NS3delN1–40, NS3delN1–70, NS3delN1–70, and NS3HadN40–70 (Fig. 4). RdRp reactions containing NS5B and either of those deletion mutants were performed. Quantification of synthesized RNA was determined by using real-time RT-PCR as described in Fig. 1. Results showed that NS3delN1–20, NS3delN1–40 and NS3HadN40–70 still stimulated NS5B RdRp activity, and the stimulations were in agreement with increasing amount of those purified deletion mutants,
Fig. 1. Effects of full-length and truncated NS3 proteins on RdRp activity of NS5B. RdRp assays containing NS5B (100 ng) and increasing amounts of NS3F(A), NS3H (B) or NS3P (C) (0, 100, 150, 200, 250,300, and 350 ng, respectively) were performed as described previously (Xiao et al., 2004). The CSFV 3 UTR (228 nt) served as a template. Quantification of synthesized RNA was determined by using real-time RT-PCR. To normalize data, the amount of CSFV RNA in each sample was divided by the amount detected in the NS5B RdRp assay in the absence of NS3 and its truncated forms under same conditions.
while NS3delN1–70 and NS3delN40–70 did not increase NS5B RdRp activity (Fig. 5 and results not shown). These results indicated that stimulations of NS5B RdRp activity by NS3 still was efficient when 20 and 40 amino acids were deleted from the N-terminal end of NS3 protease domain, respectively and implied that the 31 amino acids were from positions 40 to 70 at the N-terminal end of NS3 protease domain were important to stimulation of NS5B RdRp activity by NS3. 3.4. NS3–NS5B interaction by a 31-amino acid fragment in NS3 protease domain Furthermore, the GST-pull-down assay and the coimmunoprecipitation analysis containing NS5B and either of the
20
P. Wang et al. / Virus Research 148 (2010) 17–23
Fig. 2. GST-pull-down analysis of NS3–NS5B interactions NS3F, NS3H or NS3P was mixed with GST-NS5B, or GST (as a control), immobilized on GST resin. Each bound protein was determined by Western blot analysis with anti-NS3 monoclonal antibody. Purified NS3F, NS3H and NS3P were used as positive controls. The sizes of protein molecular mass markers are indicated on the left.
above deletion NS3 mutants was carried out. GST-pull-down assay results show that NS3delN1–20, NS3delN1–40 and NS3HadN40–70 were pulled down with NS5B (Fig. 6, lanes 3, 4 and 7), respectively, as NS3F (lane 2), but NS3delN1–70 and NS3delN40–70 were not (lanes 5 and 6), suggesting that the 31-amino acid fragment at the N-terminal end (positions 40–70) of NS3 protease domain mediated the specific NS3–NS5B interaction. Indeed, when PK-15
Fig. 4. Schematic representation of NS3 protein and its different deleted forms. Positions of amino acids (1590, 1764 and 2272) are shown over NS3 protein. NS3H and NS3P are shown down NS3 protein. The internal deletions were indicated by “V”-shape line. NS3delN1–20, NS3delN1–40, and NS3delN1–70 are the truncated NS3 proteins with deletion of 20, 40 and 70 amino acids from the N-terminal end of full-length NS3 protein, respectively. NS3delN40–70 is the truncated NS3 protein with deletion of 31 amino acids from positions 40–70 at the N-terminal end. NS3HadN40–70 is the lengthened NS3H with addition of 31 amino acids from positions 41–70 to the N-terminal end. A 31-amino acid fragment from positions 40 to 70 was shown below, and the amino acid “His (H)” was indicated by an arrow.
cells were transiently cotransfected with the pcDNA3.1 vector containing NS5B and either of the pcDNA3.1/N-FLAG vectors containing those NS3 deletion mutants, respectively, CSFV NS5B was observed to interact with NS3delN1–20, NS3delN1–40 and NS3HadN40–70 in PK-15 cells (Fig. 7, lanes 4, 6, 12), in which, CSFV NS5B did not interacted with NS3delN1–70 and NS3delN40–70 (lanes 8 and 10). Those data indicated that CSFV NS3 was bound to NS5B by the 31-amino acid fragment located in the NS3 protease domain.
Fig. 3. Co-immunoprecipitation analysis of NS3–NS5B interactions PK-15 cells were cotransfected with the pcDNA3.1 vector containing NS5B and either of the pcDNA3.1/NFLAG vector containing NS3F (lane 2), NS3H (lane 4), and NS3P (lane 6). As a control, PK-15 cells were also transfected only with the pcDNA3.1/N-FLAG vector containing NS3F (lane 1), NS3H (lane 3), or NS3P (lane 5). (A) Total lysates were fractionated by SDS-PAGE (10%) and subjected to Western blot analysis with anti-FLAG. (B) In vivo co-immunoprecipitated of NSF, NS3H and NS3P with NS5B was probed with anti-FLAG antibody (upper panel). Efficient immunoprecipitation of NS5B was also shown (lower panel).
P. Wang et al. / Virus Research 148 (2010) 17–23
21
Fig. 6. GST-pull-down analysis of different deleted NS3–NS5B interactions NS3delN1–20, NS3delN1–40, NS3delN1–70, NS3delN40–70 or NS3HadN40–70 was mixed with GST-NS5B, or GST (as a control), immobilized on GST resin. Each bound protein was determined by Western blot analysis with anti-NS3 monoclonal antibody. Purified NS3delN1–20, NS3delN1–40, NS3delN1–70, NS3delN40–70 and NS3HadN40–70 were used as positive controls. The sizes of protein molecular mass markers are indicated on the left.
Fig. 5. Effects of different deleted NS3 proteins on RdRp activity of NS5B RdRp assays containing NS5B (100 ng) and increasing amounts of NS3delN1–20 (A), NS3delN1–40 (B), or NS3HadN40–70 (C) (0, 100, 150, 200, 250,300, and 350 ng, respectively) were performed as described in Fig. 1.
4. Discussion For HCV, there is a controversy on the role of NS3 in enhancing NS5B RdRp activity. One observed that the HCV NS3 protein enhances NS5B RdRp activity (Piccininni et al., 2002). In contrast, another found that the full-length HCV NS3 does not increase NS5B RdRp activity (Zhang et al., 2005). To investigate the effect of NS3 on CSFV NS5B RdRp activity, RdRp reactions containing NS5B and NS3 proteins were performed. The CSFV 3 UTR was served as a template so that experiment results can reveal actual situation more authentically. We found that the full-length NS3 stimulated NS5B RdRp activity in a dose-dependent manner, but the truncated NS3 protein (NS3H) with helicase domain and another truncated NS3 protein (NS3P) with NS3 protease domain did not (Fig. 1). About the role of CSFV NS3 in RNA synthesis, our results are consistent
Fig. 7. Co-immunoprecipitation analysis of deletion NS3 mutant-NS5B interactions PK-15 cells were cotransfected with the pcDNA3.1 vector containing NS5B and either of the pcDNA3.1/N-FLAG vector containing NS3F (lane 2), NS3delN1–20 (lane 4), NS3delN1–40 (lane 6), NS3delN1–70 (lane 8), NS3delN40–70 (lane 10) or NS3HadN40–70 (lane 12). As a control, PK-15 cells were also transfected only with the pcDNA3.1/N-FLAG vector containing NS3F (lane 1), NS3delN1–20 (lane 3), NS3delN1–40 (lane 5), NS3delN1–70 (lane 7), NS3delN40–70 (lane 9) or NS3HadN40–70 (lane 11). The sizes of protein molecular mass markers are indicated on the left. Total lysates were fractionated by SDS-PAGE (10%) and subjected to Western blot analysis with anti-FLAG (upper panel). In vivo co-immunoprecipitated of NS3F, NS3delN1–20, NS3delN1–40, NS3delN1–70, NS3delN40–70, and NS3HadN40–70 with NS5B was probed with anti-FLAG antibody (middle panel). Efficient immunoprecipitation of NS5B was also shown (lower panel).
22
P. Wang et al. / Virus Research 148 (2010) 17–23
with those reported previously for HCV (Piccininni et al., 2002). The immunoprecipitation results, together with those of the GSTpull-down assays, show that CSFV NS5B is able to interact with either of the full-length NS3 and the NS3 protease domain, and not interact with NS3H (Figs. 2 and 3), implying that stimulation of NS5B RdRp activity is obtained by CSFV NS3 binding to the NS5B through the NS3 protease domain. It has been reported that the HCV NS3 protease domain is required for specific HCV NS3 and NS5B interaction (Zhang et al., 2005), and HCV NS5B forms a complex with NS3 through an amino-terminal portion of NS3 (Ishido et al., 1998). The interaction might up-regulate HCV NS3 helicase activity (Wen et al., 2009; Zhang et al., 2005). Our previous reports suggested that CSFV NS5B significantly enhanced the stimulative effect of NS3 on both IRES-mediated and cellular translation via the protease domain (Xiao et al., 2008). Our present data show that CSFV NS3 forms a complex with NS5B through NS3 protease domain and stimulates NS5B activity. Recent evidence has indicated that CSFV NS5B, in combination with NS3, functions in viral replication. It has been reported that rescue of virus and recover of viral RNA synthesis are not observed in the complementation experiments including either CSFV NS5B mutant or NS3 mutant (Sheng et al., 2010), suggesting that the functions of these proteins of CSFV might be cis. Both CSFV NS5B and NS3 proteins have been shown to preferentially bind to the authentic viral 3 UTR (Xiao et al., 2004; Sheng et al., 2007). Considering the fact that BVDV NS5B and NS3 have been demonstrated to be membrane-associated, with a key role for formation of the replication-transcription complex (Lai et al., 1999; Zhang et al., 2003), both CSFV NS5B and NS3 might also be membrane-associated. The fact that neither NS3H nor NS3P alone stimulate NS5B RdRp activity indicated that the full-length NS3 is required for stimulation of NS5B RdRp activity. To stimulate NS5B RdRp activity, NS3P was served as a NS5B-binding site. Furthermore, to map important regions of NS3 protease domain for stimulation of NS5B RdRp activity, a series of deletion were introduced into the NS3 protease domain (Fig. 4). The data from RdRp reactions, GST-pull-down assays and coimmunoprecipitation analysis indicated that a 31-amino acid fragment from positions 40 to 70 at the terminal end of NS3 protease domain is important for stimulation of NS5B RdRp activity and NS3–NS5B interaction (Figs. 5–7). Indeed, the truncated NS3 mutant (NS3HadN40–70) only consisting of NS3H and the 31-amino acid fragment still enhances NS5B RdRp activity (Figs. 4 and 5). Moreover, the NS5B-binding ability of the NS3HadN40–70 observed in the GST-pull assays and coimmunoprecipitation analyses is well correlated with its positive effect on NS5B RdRp activity (Figs. 5–7), indicating that CSFV NS3 stimulates NS5B RdRp activity by binding directly to NS5B through the 31-amino acid fragment located in the NS3 protease domain. We analyzed the amino acid composition of the fragment. It was found that there were 20 polar and hydrophilic ones among 31 amino acids, which is one of the properties of protein binding sites because protein–protein interfaces are often studded with many polar residues or hydrophilic residues (Hu et al., 2000; Xu et al., 1997b,c). Moreover, at the interface, there are higher proportions of buried charged and polar residues as compared to protein cores, suggesting that hydrogen bonds and ion pairs contribute to the stability of protein binding (Keskin et al., 2008). Furthermore, by an alignment analysis we found that the conserved amino acid “His (H)” at position 39 was located in the 31-amino acid fragment (Fig. 4 and results not shown). It is one of those residues predicted to form the classical serine protease catalytic triad (Ryan et al., 1998), which might also be important for a protein binding site. Although our results about the role of CSFV NS3 in RNA synthesis are consistent with those reported for HCV in the paper of Piccininni et al. (2002), our present conclusion that CSFV NS3 RNA helicase does not increase NS5B RdRp activity is contrary to that
from the paper, in which, HCV NS3 helicase domain has been found to alone enhance RNA synthesis. We speculate that the difference is due to virus species, but without experimental evidence support the proposal. Although HCV NS3 RNA helicase is necessary for viral replication (Lam and Frick, 2006), the NS3 protease domain is also important to RNA helicase. It has been found in HCV that NS3 protease domain is required for RNA unwinding by NS3, and when the NS3 protein lacks the protease domain it unwinds RNA more slowly (Beran et al., 2007; Frick et al., 2004). Therefore, a fulllength NS3 protein might be important not only for NS5B RdRp activity and also for NS3 RNA helicase in HCV and CSFV. The role of NS3 in the cytopathogenicity and life cycle of CSFV is also important. The cytopathogenicity of pestiviruses including CSFV has been demonstrated to correlate with the expression of large amounts of NS3 (Aoki et al., 2004; Gallei et al., 2008; Meyers and Thiel, 1995). Therefore, NS3 is regarded as marker protein for cytopathogenic pestiviruses. The data from complementation experiments have showed that NS2–3 and NS3, each in association with NS4A, have independent functions in the CSFV life cycle (Moulin et al., 2007). An essential function for NS3 in pestiviral RNA replication which cannot be supplied by its NS2–3 precursor has been implied in BVDV replication analysis (Lackner et al., 2004). Taken together, we found that CSFV NS3 protease is the place to mediate a NS3–NS5B interaction for stimulation of NS5B RdRp activity. The 31-amino acid fragment localized at positions 40–70 of the NS3 protease might be the NS5B-binding site. This is important for understanding the mechanism for CSFV replication. However, which region of NS5B is the NS3-binding site remains to be elucidated. Acknowledgements This work was supported by the National Natural Science Foundation of China (30670445, 30870492), the Shanghai Municipal Science and Technology Commission (07DZ12038, 08JC1416900), the Shanghai Municipal Education Commission (08ZZ66), the Shanghai Leading Academic Discipline Project (S30406) and the Leading Academic Discipline Project of Shanghai Normal University (DZL709). References Aoki, H., Sakoda, Y., Nakamura, S., Suzuki, S., Fukusho, A., 2004. Cytopathogenicity of classical swine fever viruses that do not show the exaltation of Newcastle disease virus is associated with accumulation of NS3 in serumfree cultured cell lines. J. Vet. Med. Sci. 66, 161–167. Becher, P., Thiel, H.-J., 2002. Genus Pestivirus (Flaviviridae). In: Tidona, C.A., Darai, G. (Eds.), The Springer Index of Viruses. Springer-Verlag, Heidelberg, Germany, pp. 327–331. Beran, R.K., Serebrov, V., Pyle, A.M., 2007. The serine protease domain of hepatitis C viral NS3 activates RNA helicase activity by promoting the binding of RNA substrate. J. Biol. Chem. 282, 34913–34920. Cuthbert, J.A., 1994. Hepatitis C: progress and problems. Clin. Microbiol. Rev. 7, 505–532. Dimitrova, M., Imbert, I., Kieny, M.P., Schuster, C., 2003. Protein–protein interactions between hepatitis C virus nonstructural proteins. J. Virol. 77, 5401–5414. Frick, D.N., Rypma, R.S., Lam, A.M., Gu, B., 2004. The nonstructural protein 3 protease/helicase requires an intact protease domain to unwind duplex RNA efficiently. J. Biol. Chem. 279, 1269–1280. Gallei, A., Blome, S., Gilgenbach, S., Tautz, N., Moennig, V., Becher, P., 2008. Cytopathogenicity of classical swine fever virus correlates with attenuation in the natural host. J. Virol. 82, 9717–9729. Gong, Y., Trowbridge, R., Macnaughton, T.B., Westaway, E.G., Shannon, A.D., Gowans, E.J., 1996. Characterization of RNA synthesis during a one-step growth curve and of the replication mechanism of bovine viral diarrhea virus. J. Gen. Virol. 77, 2729–2736. Gu, B., Liu, C., Lin-Goerke, J., Maley, D.R., Gutshall, L.L., Feltenberger, C.A., Del Vecchio, A.M., 2000. The RNA helicase and nucleotide triphosphatase activities of the bovine viral diarrhea virus NS3 protein are essential for viral replication. J. Virol. 74, 1794–1800. Heinz, F.X., Collett, M.S., Purcell, R.H., Gould, E.A., Howard, C.R., Houghton, M., Moormann, R.J.M., Rice, C.M., Thiel, H.-J., 2000. Family Flaviviridae. In: Fauquet, C.M., van Regenmortel, M.H.V., Bishop, D.H.L., Carstens, E.B., Estes, M.K., Lemon, S.M.,
P. Wang et al. / Virus Research 148 (2010) 17–23 Maniloff, J., Mayo, M.A., McGeoch, D.J., Pringle, C.R., Wickner, R.B. (Eds.), Virus Taxonomy: Seventh Report of the International Committee on Taxonomy of Viruses. Academic Press, Inc., San Diego, CA, pp. 859–878. Hu, Z., Ma, Buyong, Wolfson, H., Nussinov, R., 2000. Conservation of polar residues as hot spots at protein interfaces. Proteins: Struct. Funct. Bioinform. 39, 331–342. Ishido, S., Fujita, T., Hotta, H., 1998. Complex formation of NS5B with NS3 and NS4A proteins of hepatitis C virus. Biochem. Biophys. Res. Commun. 244, 35–40. Lam, A.M., Frick, D.N., 2006. Hepatitis C virus subgenomic replicon requires an active NS3 RNA helicase. J. Virol. 80, 404–411. Keskin, O., Gursoy, A., Ma, B., Nussinov, R., 2008. Principles of protein–protein interactions: what are the preferred ways for proteins to interact? Chem. Rev. 108, 1225–1244. Kolykhalov, A.A., Mihalik, K., Feinstone, S.M., Rice, C.M., 2000. Hepatitis C virus-encoded enzymatic activities and conversed RNA elements in the 3 nontranslated region are essential for virus replication in vivo. J. Virol. 74, 2046–2051. Lackner, T., Müller, A., Pankraz, A., Becher, P., Thiel, H.J., Gorbalenya, A.E., Tautz, N., 2004. Temporal modulation of an autoprotease is crucial for replication and pathogenicity of an RNA virus. J. Virol. 78, 10765–10775. Lai, V.C.H., Kao, C.C., Ferari, E., Park, J., Uss, A.S., Wright-Minogue, J., Hong, Z., Lai, J.Y.N., 1999. Mutational analysis of bovine viral diarrhea virus RNA-dependent RNA polymerase. J. Virol. 73, 10129–10136. Lindströma, H., Lundina, M., Häggströma, S., Persson, M.A.A., 2006. Mutations of the hepatitis C virus protein NS4B on either side of the ER membrane affect the efficiency of subgenomic replicons. Virus Res. 121, 169–178. Lundin, M., Monne, M., Widell, A., von Heijne, G., Persson, M.A.A., 2003. Topology of the membrane-associated hepatitis C virus protein NS4B. J. Virol. 77, 5428–5438. Masaki, T., Suzuki, R., Murakami, K., Aizaki, H., Ishii, K., Murayama, A., Date, T., Matsuura, Y., Miyamura, T., Wakita, T., 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. Meyers, G., Thiel, H.J., 1995. Cytopathogenicity of classical swine fever virus caused by defective interfering particles. J. Virol. 69, 3683–3689. Moennig, V., Plagemann, P.G.W., 1992. The pestiviruses. Adv. Virus Res. 41, 53–98. Moulin, H.R., Seuberlich, T., Bauhofer, O., Bennett, L.C., Tratschin, J.D., Hofmann, M.A., Ruggli, N., 2007. Nonstructural proteins NS2–3 and NS4A of classical swine fever virus: essential features for infectious particle formation. Virology 365, 376– 389. Piccininni, S., Varaklioti, A., Nardelli, M., Dave, B., Raney, K.D., McCarthy, J.E., 2002. Modulation of the hepatitis C virus RNA-dependent RNA polymerase activity by the non-structural (NS) 3 helicase and the NS4B membrane protein. J. Biol. Chem. 277, 45670–45679. Ryan, M.D., Monaghan, S., Flint, M., 1998. Virus-encoded proteinases of the Flaviviridae. J. Gen. Virol. 79, 947–959. Sheng, C., Xiao, M., Geng, X., Liu, J., Wang, Y., Gu, F., 2007. Characterization of interaction of classical swine fever virus NS3 helicase with 3 untranslated region. Virus Res. 129, 43–53. Sheng, C., Zhua, Z., Yua, J., Wan, L., Wang, Y., Chen, J., Gu, F., Xiao, M., 2010. Characterization of NS3, NS5A and NS5B of classical swine fever virus through mutation and complementation analysis. Vet. Microbiol. 140, 72–80.
23
Steffen, S., Thiel, H.J., Behrens, S.E., 1999. The RNA-dependent RNA polymerase of different members of the family Flaviviridae exhibits similar properties in vitro. J. Gen. Virol. 80, 2583–2590. Suzich, J.A., Tamura, J.K., Palmer-Hill, F., Warrener, P., Grakoui, A., Rice, C.M., Feinstone, S.M., Collett, M.S., 1993. Hepatitis C virus NS3 protein polynucleotidestimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes. J. Virol. 67, 6152–6158. Tellinghuisen, T.L., Marcotrigiano, J., Gorbalenya, A.E., Rice, C.M., 2004. The NS5A protein of hepatitis C virus is a zinc metalloprotein. J. Biol. Chem. 279, 48576–48587. Tellinghuisen, T.L., Paulson, M.S., Rice, C.M., 2006. The NS5A protein of bovine viral diarrhea virus contains an essential zinc-binding site similar to that of the hepatitis C virus NS5A protein. J. Virol. 80, 7450–7458. Wang, Y., Xiao, M., Chen, Jun., Zhang, W., Luo, J., Bao, K., Nie, M., Chen, J., Li, Bo., 2007. Mutational analysis of the GDD sequence motif of classical swine fever virus RNA-dependent RNA polymerases. Virus Genes 34, 63–65. Warrener, P., Collett, M.S., 1995. Pestivirus NS3 (p80) protein possesses helicase activity. J. Virol. 69, 1720–1726. Wen, G., Xue, J., Shen, Y., Zhang, C., Pan, Z., 2009. Characterization of classical swine fever virus (CSFV) nonstructural protein 3 (NS3) helicase activity and its modulation by CSFV RNA-dependent RNA polymerase. Virus Res. 141, 63–70. Xiao, M., Chen, J., Li, B., 2003. RNA-dependent RNA polymerase activity of classical swine fever virus NS5B protein expressed in natural host cells. Acta Virol. 47, 17–26. Xiao, M., Zhang, C.Y., Pan, Z.S., Wu, H.X., Guo, J.Q., 2002. Classical swine fever virus NS5B-GFP fusion possesses an RNA-dependent RNA polymerase activity. Arch. Virol. 147, 1779–1785. Xiao, M., Gao, J., Wang, W., Wang, Y., Chen, J., Chen, J., Li, B., 2004. Specific interaction between the classical swine fever virus NS5B protein and the viral genome. Eur. J. Biochem. 271, 3888–3896. Xiao, M., Li, H., Wang, Y., Wang, X., Wang, W., Peng, J., Chen, J., Li, B., 2006. Characterization of the N-terminal domain of classical swine fever virus RNA-dependent RNA polymeras. J. Gen. Virol. 87, 347–356. Xiao, M., Bai, Y., Xu, H., Geng, X., Chen, J., Wang, Y., Chen, J., Li, B., 2008. Effect of NS3 and NS5B protein on classical swine Fever Virus IRES-mediated translation and its host cellular translation. J. Gen. Virol. 89, 994–999. Xu, J., Mendez, E., Caron, P.R., Lin, C., Murcko, M.A., Collett, M.C., Rice, C.M., 1997a. Bovine viral diarrhea virus: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication. J. Virol. 71, 5312–5322. Xu, D., Lin, S.L., Nussinov, R., 1997b. Protein–protein interfaces are studded with many polar residues or hydrophilic residues. J. Mol. Biol. 265, 68–84. Xu, D., Tsai, C.J., Nussinov, R., 1997c. Hydrogen bonds and salt bridges across protein–protein interfaces. Protein Eng. 10, 999–1012. Zhang, C., Kim, Y.C., Kumar, R., Yuan, F., Shi, P.Y., Kao, C., Luo, G., 2005. Stimulation of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase activity by the NS3 protease domain and by HCV RNA-dependent RNA polymerase. J. Virol. 79, 8687–8697. Zhang, G., Flick-Smith, H., McCauley, J.W., 2003. Differences in membrane association and sub-cellular distribution between NS2–3 and NS3 of bovine viral diarrhoea virus. Virus Res. 97, 89–102.