Characterization of interaction of classical swine fever virus NS3 helicase with 3′ untranslated region

Virus Research 129 (2007) 43–53

Characterization of interaction of classical swine fever virus NS3 helicase with 3 untranslated region Chun Sheng a,b , Ming Xiao a,∗ , Xiaolu Geng a , Jiaying Liu a , Yujing Wang a , Fukang Gu b a

College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, China b School of Life Science, East China Normal University, Shanghai 200062, China Received 4 February 2007; received in revised form 3 May 2007; accepted 4 May 2007 Available online 12 June 2007

Abstract The classical swine fever virus (CSFV) full-length NS3 protein (NS3F) and the truncated NS3 protein (NS3H) with postulated helicase domain were expressed and demonstrated to have helicase activity. Further, the electrophoretic mobility shift assays containing NS3H and the viral 3 terminal sequences showed that NS3H specifically bound to the plus- and minus-strand 3 UTR. The minus-strand 3 UTR had higher binding activity. The 21-nt fragments at the 3 -most terminal sequences of both 3 UTRs were essential to NS3H binding. A 12-nt insertion, CUUUUUUCUUUU, present in the 3 UTR of a CSFV live attenuated vaccine strain, was also found to be deleterious to helicase binding. Intact secondary structure of 3 terminal sequence of 3 UTR might be important in helicase binding. Our results show that interaction between the helicase and the viral 3 UTR is similar to that between the replicase and the 3 UTR, suggesting that NS3 helicase is important for CSFV genomic replication. © 2007 Elsevier B.V. All rights reserved. Keywords: Classical swine fever virus; NS3; Helicase; 3 UTR

1. Introduction Classical swine fever virus (CSFV) is the causative agent of classical swine fever, a highly contagious and sometimes fatal viral disease of pigs, and can cause a considerable economic loss. The Pestivirus genus within the family Flaviviridae is comprised of CSFV, bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, and border disease virus (BDV) (Becher and Thiel, 2002; Heinz et al., 2000). The hepatitis C virus (HCV), the major cause of transfusion-associated hepatitis, also belongs to this family (Cuthbert, 1994). The genome of the CSFV 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 polyprotein is processed into mature proteins by cellular as well as viral proteases: NH2 – Npro –C–Erns –E1–E2–p7–NS2–NS3–NS4A–NS4B–NS5A–NS∗ Corresponding author at: Biology Department, College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, China. Tel.: +86 21 64321022; fax: +86 21 65642468. E-mail address: [email protected] (M. Xiao).

0168-1702/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2007.05.004

5B–COOH (Moennig and Plagemann, 1992). The 3 UTR is most likely involved in initiation of the pestiviral genome replication (Isken et al., 2003, 2004; Pankraz et al., 2005; Xiao et al., 2004b; Yu et al., 1999), also involved in the coordination of the viral translation and replication (Isken et al., 2004). Moreover, the 3 UTR may contact the 5 UTR by RNA–RNA interactions (Isken et al., 2003). In addition to regulation of genome replication, the 5 UTR is able to regulate translation of the viral genomes (Fletcher and Jackson, 2002). The CSFV NS5B protein has an RNA-dependent RNA polymerase (RdRp) activity (Steffen et al., 1999; Xiao et al., 2006), contains a conserved GDD motif necessary for the catalytic activity (Jablonski et al., 1991; Lohmann et al., 1997; Lai et al., 1999). In additional to NS5B proteins, 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). 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 NS3 primarily serves as a protease to process the viral polyprotein (Xu et al., 1997). The helicase and NTPase

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activities are localized to the C-terminal of NS3 protein (Suzich et al., 1993; Warrener and Collett, 1995). The HCV and BVDV NS3 proteins have been demonstrated to have RNA helicase activity (Kim et al., 1995; Tai et al., 1996; Warrener and Collett, 1995), as that of other member under the family Flaviviridae (Li et al., 1999). Some plus-strand RNA viruses, such as poliovirus, rhinovirus, without NS3 proteins, encode a homologous protein called 2C with the helicase and NTPase activity (Pfister and Wimmer, 1999). Our bioinformatics analysis shows that the CSFV NS3 protein contains canonical amino acid motifs present in all superfamily II RNA helicases (Kadare and Haenni, 1997), therefore, the protein is postulated to have RNA helicase activity. However, more biochemical evidences are required to characterization of the CSFV NS3 protein helicase activity. In this report, we express the CSFV NS3 protein and apply competitive electrophoretic mobility shift assays (EMSA) to characterization of interaction of CSFV NS3 helicase with 3 terminal sequences of the viral genome. 2. Materials and methods 2.1. Expression and purification of NS3 proteins The recombinant plasmid was constructed for expression of CSFV NS3 protein. Total RNA was extracted from CSFV Shimen strain. A full-length NS3 cDNA encoding amino acids 1590–2272, was obtained by RT-PCR, and cloned into the pET28 (a) vectors. The cDNA of the truncated NS3 with postulated helicase domain (amino acids 1764–2272) was also cloned (Fig. 1). Additional sequences coding for six histidines at the C terminus were engineered to facilitate the purification of 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 the bacteriophage T7 RNA polymerase. Expression was induced by addition of isopropyltiogalactoside (IPTG). The bacterial cell culture was harvested by centrifugation at 6000 × g for 10 min, and washed with 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. After undergoing freezing and thawing once, cells were subject to sonication. The cleared lysate was obtained by centrifugation at 35,000 × g for 15 min. The cleared lysate containing the recombinant protein was purified using nickel–nitrilotriacetic acid (Ni–NTA)–sepharose resin (Gibco BRL). Briefly, the CSFV NS3 protein with a polyhistidine tag was bound to the Ni–NTA resin preequilibrated with the above buffer, and then washed with the buffer containing 50 mM imidazole. The bound NS3 was eluted with the buffer containing different concentrations of imidazole (100–500 mM). The NS3 protein was collected and combined and dialyzed in the buffer A (50 mM Tris–HCl [pH 8.0], 1 mM DTT, 50 mM NaCl, 5 mM MgCl2 , 10% glycerol). NS3 protein solutions and dilutions of bovine serum albumin with known concentration were subjected

Fig. 1. Schematic drawing and expression of the CSFV full-length NS3 protein (N3F) and truncated NS3 protein with helicase domain (N3H). (A) Schematic drawing of N3F and N3H. Positions of amino acids (1590, 1764 and 2272) are indicated. (B) Expression of N3F or N3H was induced by addition of isopropyltiogalactoside (IPTG). Proteins were purified using nickel–nitrilotriacetic acid (Ni–NTA) resin. The expression products were subject to separation by SDSPAGE, visualized by Coomassie blue staining. The name of expression products is shown on the top. M: the sizes of protein molecular mass markers; C: E. coli lysate as control.

to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels with the samples were stained with Coomassie brilliant blue. The amount of NS3 protein was determined by densitometry scanning and comparing the two samples on the same gel. Purified proteins were separated by SDS-PAGE. The truncated NS3 protein with postulated helicase domain was expressed and purified as described above. 2.2. RNA preparation The RNAs were generated as described previously (Xiao et al., 2004a,b). In brief, cDNA fragments containing complete CSFV 3 UTR, 5 UTR and random coding sequences were initially cloned into the pGEM-T vector (Promega) from total CSFV RNA by RT-PCR, respectively. These nucleotide sequences were verified. A pair of primers at the two sides of expectedly mutant fragment, or desired wild-type sequence was designed. The standard PCR method based on the primers was used. The PCR product was obtained, treated with E. coli Klenow, then with T4DNA ligase, cloned into the pGEM-T vector and transformed E. coli BL21 (DE3). Plasmids were extracted and sequenced. The plasmids containing expected mutation were verified by sequencing. Wild-type and mutated RNA templates were synthesized by PCR and subsequent in vitro transcription based on these RT-PCR products. A DNA Vent polymerase and the primer containing bacteriophage T7 promoter were used in the PCR. After verifying the sequence, the resulting PCR products were served as the template for

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the subsequent in vitro transcription. The in vitro transcription was performed in 50 ␮l of reaction mixtures following the standard method: 20 ␮l of 5× transcription buffer, 2 ␮l of Rnasin (20–40 U/␮l) (Promega), 5 ␮l of each NTP (2.5 mM), 5 ␮g of the template, 2 ␮l of T7 RNA polymerase (10–20 U/␮l) (Promega). The mixture was incubated at 37 ◦ C for 2 h. Ten microliters of Dnase I (Takara) was added to the mixture and incubated at 37 ◦ C for 15 min. The mixture was extracted with phenol/chloroform/isoamylalcohol. After ethanol precipitation, the RNA was dried, and redissolved in 20 ␮l of dd H2 O. Labeled RNA fragments were produced in the analogous way with [␣32 P] UTP. Integrity of the RNA was analyzed by denaturing formaldehyde–agarose gel electrophoresis. The concentration of RNA was determined by measuring its optical density at 260 nm. 2.3. RNA helicase assays RNA helicase assays were performed as previously described with the slight modifications (Warrener and Collett, 1995). The 3 terminal sequence of plus-strand RNA of the viral genome (longer strand) and its partial complementary strand (shorter strand) formed the substrate for RNA helicase assays. A 168-nt longer strand RNA and a 40 shorter strand RNA were prepared by in vitro transcription as described in the above section of RNA preparation. The shorter strand RNA was transcribed in the presence of [␣-32 P] UTP and radiolabeled. Both RNAs were combined at a molar ratio of the shorter strand to the longer strand of approximately 5:1 in a hybridization buffer solution containing 0.5 M NaCl, 25 mM HEPES (pH 7.4), 1 mM EDTA, 0.1% sodium dodecyl sulfate (SDS). The mixture was heated for 5 min at 95 ◦ C and 30 min at 65 ◦ C and then incubated overnight at 25 ◦ C. The generated partial duplex RNA substrates were electrophoresed on a 6% nondenaturing polyacrylamide gel, eluted with 400 ␮l of elution buffer (0.6 M ammonium acetate, 1 mM EDTA, 0.2% SDS) from the gel, and then purified. Helicase activity was assayed in 20 ␮l reaction mixture containing 25 mM MOPS-KOH (pH 6.5), 3 mM MgCl2 , 2 mM DTT, 0.01% of BSA, 1 U of RNasin (Promega), 400 ng CSFV NS3 helicase, and 0.25 nM 32 P-labeled partial duplex RNA substrate. After preincubation for 15 min at room temperature, 5 mM ATP was added to start the helicase reaction. The reaction mixtures were further incubated for 30 min at 37 ◦ C, and then stopped by adding 5 ␮l of 5× termination buffer (0.1 M Tris–HCl, pH 7.4, 20 mM EDTA, 0.5% SDS, 0.1% NP40, 0.1% xylene cyanol, 0.1% bromophenol blue, 50% glycerol). The reaction products were electrophoresed on 6% polyacrylamide gel. The gel was dried and then visualized by autoradiography.

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plus-strand or minus-strand genome was used as the probe. The reactions were performed at room temperature for 30 min. The reaction products were analyzed on 6% polyacrylamide gel. The gel was dried and subjected to autoradiography. 2.5. Prediction of secondary structures The soft ware for prediction of secondary structures of RNA templates is RNAstructure 3.2. Maximum % energy difference, maximum number of structures, and window size are designed as 10, 20 and 5, respectively. The cDNA sequence of RNA template was used. 3. Results 3.1. The CSFV NS3 protein possesses RNA helicase activity The HCV, BVDV and Dengue Virus NS3 proteins have been demonstrated to have RNA helicase activity (Kim et al., 1995; Li et al., 1999; Tai et al., 1996; Warrener and Collett, 1995). The alignment analysis shows that the CSFV NS3 protein contains canonical amino acid motifs present in all superfamily II RNA helicases and the protein is postulated to have RNA helicase activity. To demonstrate the CSFV NS3 protein to indeed possess RNA helicases, the full-length NS3 protein (NS3F) and the truncated NS3 protein with the postulated helicase domain (NS3H) were expressed in E. coli (Fig. 1), then subjected to

2.4. Competitive electrophoretic mobility shift assays In each competitive electrophoretic mobility shift assays (EMSA), unless otherwise specified, 1 pmol of labeled RNA was incubated with 450 ng of NS3H in a buffer containing 20 mM Hepes (pH 7.3), 5 mM MgCl2 , 7.5 mM DTT, 5% glycerol, 125 mM NaCl, 100 ␮g of bovine serum albumin per ml, 1 U of RNasin (Promega), and various amount of competitor RNA. The [␣-32 P] UTP-labeled RNA fragment containing 3 UTR of

Fig. 2. RNA unwinding activity of NS3F or NS3H. A 168-nt longer strand RNA and a 40 shorter strand RNA were prepared by in vitro transcription as described in Section 2. The shorter strand RNA was radiolabeled by [␣-32 P] UTP. Both RNAs were combined into partial duplex RNA, served as the substrates for helicase assays. Helicase activity was assayed as described in Section 2. The reaction products were electrophoresed on 6% polyacrylamide gel. The gel was dried and then visualized by autoradiography. The name of the protein taking part in RNA unwinding reaction is shown on the top. The RNA unwinding reaction without NS3F or NS3H is indicated with “C”.

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Fig. 3. NS3 helicase binds to 3 terminal sequences of CSFV plus-strand RNA. (A) Increasing enzyme concentrations of the NS3H (lanes 2–7, 80, 150, 250, 350, 450 and 550 ng) were added in a standard EMSA containing a fixed amount of the radiolabeled PT0. Lane 1 represents for free PT0 probe to which no NS3H was added. (B) The EMSA was done under the condition of a fixed amount of the NS3H in the presence of increasing amount of competitor PT0 (lanes 1–7, 0, 1, 5, 10, 25, 50 and 100 pmol). The names of competitors were indicated on the top. The arrowheads represent the orientation for increasing amount. The reactions were performed in a buffer containing 20 mM Hepes (pH 7.3), 5 mM MgCl2 , 7.5 mM DTT, 5% glycerol, 125 mM NaCl, 100 ␮g of bovine serum albumin per ml, 1 U of RNasin (Promega) at room temperature for 30 min. The reaction products were analyzed on 6% polyacrylamide gel. The gel was dried and subjected to autoradiography.

the RNA helicase assays. The experiment showed that both the NS3F and the NS3H have RNA helicase activity, as expected (Fig. 2). It was observed that the NS3F had higher helicase activity than NS3H, consistent with the recent report, in which the helicase activity of HCV full-length NS3 protein was higher than that of the truncated NS3 protein with helicase domain (Zhang et al., 2005). The possible reason is that the protease domain of the NS3 protein up-regulates the helicase activity of the helicase domain. 3.2. NS3 helicase binds to 3 terminal sequences of CSFV plus-strand RNA Many single plus-strand RNA virus helicases have been shown to specifically interact with the 3 terminal sequences of viral genome (Banerjee et al., 1997; Banerjee and Dasgupta, 2001). To characterize the interaction of CSFV NS3 helicase with viral genome, we applied the competitive electrophoretic mobility shift assays (EMSA) to analysis of binding ability of the NS3 helicase to different RNA templates. The 3 UTR of CSFV plus-strand RNA is believed to be the first entry site for viral replicases to initiate RNA genome replication. The helicase activity is needed to unwind the secondary structure present in the 3 UTR. Therefore, we first examined the binding of the NS3H to the 3 UTR. A 3 terminal sequence of the CSFV plus-strand genome, designed as PT0, was produced and labeled with [␣-32 P] UTP. The purified NS3H at various concentrations

were incubated with a fixed amount of the radio-labeled PT0. As shown in Fig. 3A, the amount of RNA–NS3H complex retarded at the loading wells was in agreement with increasing amount of purified NS3H. At the same time, the EMSA was done under the condition of a fixed amount of the NS3H and the radio-labeled PT0 in the presence of increasing amount of competitor, the unlabeled PT0. The EMSA showed that RNA–NS3H complex could be specifically competed by the unlabeled PT0. These results clearly show that the NS3H are capable of interacting with PT0. 3.3. Mapping of the NS3H-binding site on the CSFV genome To determine if the NS3H interacting with the 3 terminal sequence specifically binds to 3 UTR, competitive binding assays were performed with different RNA templates of CSFV genome. These RNA templates are the plus-strand RNA competitors, such as PT0 (a 603-nt RNA template containing the full-length 228-nt 3 UTR and the subsequent 375-nt coding sequence), PTr1 (a 375-nt RNA fragment corresponding to the coding sequence next to 3 UTR) (Fig. 4A), +5 UTR (5 UTR of the plus-strand genome), PTr2 (a random sequence corresponding to positions 1245–1700 of the plus-strand genome). These templates were, respectively, added to the binding reaction containing NS3H and radio-labeled PT0. The results showed that the radio-labeled PT0–NS3H complex was almost com-

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Fig. 4. Mapping of the NS3H-binding site on the CSFV genome. (A) Schematic drawing of the partial RNA templates used for mapping of the NS3H-binding site on the CSFV genome. PT0, a 603-nt RNA template containing the full-length 228-nt 3 UTR and the subsequent 375-nt coding sequence; PTr1, a 375-nt RNA fragment corresponding to the coding sequence next to 3 UTR; MT0, a 701-nt fragment containing the full-length 373-nt minus-strand 3 UTR and the subsequent 328-nt upstream sequence; MTr1, a 328-nt fragment next to minus-strand 3 UTR. (B) The competitive EMSA was preformed with the NS3H and the 32 P-labeled PT0 in the presence (lanes 3–14) of increasing amount (1, 5 and 25 pmol) of various competitors (PT0, +5 UTR, PTr1, and PTr2). Lane 1 represents for free PT0 probe to which no NS3H was added, lane 2 for without competitors. (C) The results were obtained from the competitive EMSA with NS3H containing unlabeled PT0 (lanes 6 to 8, 10, 25 and 50 pmol) or MT0 (lanes 3–5, 10, 25 and 50 pmol) in presence of the 32 P-labeled MT0. Lanes 1 and 2 represents for no unlabeled MT0 and no NS3H, respectively.

pletely abolished by 25-fold molar excess of the unlabeled PT0 (Fig. 4B, lane 5). Other RNA templates were very poor competitors (Fig. 4B, lanes 6–14). In same way, the minus RNA templates of the CSFV genome were included in the competitive EMSA. These templates were MT0 (a 701-nt fragment containing the full-length 373-nt minus-strand 3 UTR and the subsequent 328-nt upstream sequence), MTr1 (a 328-nt fragment next to minus-strand 3 UTR) (Fig. 4A), MTr2 (a random sequence corresponding to positions 2033–2500 of the minusstrand genome) and −5 UTR (5 UTR of the minus strand). As expected, the MT0 competed most effectively with the labeled RNA, and that MTr1, MTr2 and −5 UTR had no effect at all (results not shown). Taken together, these results suggest that the NS3H specifically binds to the plus-strand or the minus-strand 3 UTR of CSFV genome. It is known that both the plus genomic and the minus genomic RNAs are present in infected host cells when the replication of RNA virus occurs. For comparison between plus-strand 3 UTR and the minus-strand 3 UTR in NS3H binding, the purified NS3H was incubated with the radio-labeled MT0 in the presence of the unlabeled PT0 or the unlabeled MT0. Interestingly, the MT0 was stronger than the PT0 in the interaction with the NS3H (Fig. 4C). The purified NS3H was incubated with the radio-

labeled PT0 in the presence of the unlabeled PT0 or the unlabeled MT0. The same results were obtained (results not shown). These data indicated that the binding of NS3H to the minus-strand 3 UTR was more efficient to the plus-strand 3 UTR. 3.4. Effects of 3 terminal sequences of 3 UTR on helicase binding Influence of 3 terminal sequences of 3 UTR on helicase binding was examined by the EMSA containing 3 terminal sequences of 3 UTR mutants. The plus-strand 3 UTR mutants, PT13, PT14, PT15, PT16, PT17, were produced. They represent the mutants with deletion of 3 C, 3 CC, 3 CCC, 3 CCCGG and a 21-nt fragment at the 3 -most terminal sequence of plusstrand 3 UTR, respectively (Fig. 5). The radio-labeled PT0 was used as the substrate for the NS3H under the condition for the competitive experiment. Among these RNA templates, PT13 competed most efficiently (Fig. 6A, lanes 2–4), followed by PT14 (lanes 5–7), PT15, PT16 and PT17 were defective (lanes 8–16). These results indicated that the CSFV NS3H binds to the 3 UTR mainly through interaction with the approximate 3 -most 21 nucleotide acids. The 3 CCCGG and 3 CCC at 3 terminal of the plus-strand 3 UTR were also important for

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Fig. 5. Schematic drawing of the RNA templates used for characterization of interaction between the NS3 helicase and the 3 terminal sequences of CSFV genome. The name of RNA template is indicated on the left. PT0 to PT17 represents the 3 terminal sequences of plus-strand RNA. MT0 to MT5 is the 3 terminal sequences of minus-strand RNA. S3 and H3 are the 3 terminal sequences of Shimen strain and HCLV strain, respectively. H3  is the HCLV 3 terminal sequence with deletion of the 12-nt insertion (CUUUUUUCUUUU), and S3 + is the Shimen 3 terminal sequence with the 12-nt insertion. The 12-nt insertion was continuously underlined. The suspension points (. . .) represent the other part of templates. The single underlined nucleotides are introduced by mutagenesis. N stands for the deleted nucleotide in templates. The numbers represent the positions of neighboring nucleotides in 3 UTRs. The positions of 5 -most terminal nucleotide of these 3 UTRs are designed 1. The plus-strand and minus-strand 3 UTRs of CSFV Shimen contain, respectively, 228 nucleotides (positions 1–228) and 373 nucleotides (positions 1–373).

NS3H binding. Similarly, the binding experiment was performed towards the minus-strand 3 UTR mutants, MT1 to MT5, representing, respectively, the RNA templates with deletion of 3 C, 3 CAUAUG, 3 CAUAUGCU, 3 CAUAUGCUC and 21-nt fragment at the 3 -most terminal sequence of the minus-strand 3 UTR (Fig. 5). It was found that the MT1 (Fig. 6B, lanes 2–4) was the most efficient competitor whereas MT4 and MT5 was the poorest (lanes 11–16), indicating that the 3 CATATGCTC sequence and 21-nt fragment of 3 terminal of the minus-strand strand genome is essential to NS3H binding.

We previously found that some mutations within the 21-nt terminal sequence of plus-strand 3 UTR were deleterious to initiation of RNA synthesis by RdRp (Xiao et al., 2004c). To determine if the mutations are also deleterious to NS3H binding, we also applied EMSA experiments to the mutants. In our previous experiments, the nucleotide at position 216 of the 3 UTR was most important, that at position 219 also important, and that at position 212 most unimportant, to initiation of RNA synthesis (Xiao et al., 2002, 2004c). Therefore, the nucleotides at positions 212, 216 and 219 were selected as our target for mutagene-

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Fig. 6. Effects of 3 terminal sequences of 3 UTR on helicase binding. (A) The competitive EMSA was preformed with the NS3H and the 32 P-labeled PT0 in absence (lane 1) or the presence (lanes 2–16) of increasing amount (10, 25 and 50 pmol) of various RNA templates with deletion of plus 3 terminal sequences (PT13–PT17). (B) The competitive EMSA was preformed with the NS3H and the 32 P-labeled MT0 in absence (lane 1) or the presence (lanes 2–16) of increasing amount (10, 25 and 50 pmol) of various RNA templates with deletion minus 3 terminal sequences (MT1–MT5).

sis. Each nucleotide was replaced by other three nucleotides, respectively, or was deleted. Together, 12 mutants (PT1–PT12) were formed (Fig. 5). Results showed that almost all replacements were non-deleterious to the binding activity of 3 terminal sequence. Only deletion mutation slightly reduced the binding (results not shown), indicating that individual nucleotides within the 21-nt terminal sequence might be unimportant to NS3 helicase binding. 3.5. Effects of a 12-nt insertion present in the 3 UTR on helicase binding The CSFV HCLV strain, a live attenuated vaccine strain, was obtained from the CSFV Shimen strain after 480 passages in the rabbit in China in the 1950s (Wu et al., 2001), was considered to be one of the most effective and safest live vaccine (Moormann et al., 1996). An additional insertion of 12 continuous nucleotides, CUUUUUUCUUUU, is detected in the 3 UTR of HCLV genome, compared with its parental virulent Shimen strain (Bj¨orklund et al., 1998; Wu et al., 2001). Our previous reports have shown that the 12-nt insertion resulted in reduction in RNA synthesis, therefore, might be one of the reasons for avirulence (Xiao et al., 2004a). To examine if the 12-nt insertion has an effect on NS3 helicase binding, we compared the binding activity of Shimen 3 UTR (S3 ) with that of HCLV 3 UTR (H3 ) by competitive EMSA. As shown in Fig. 7, the HCLV 3 UTR is indeed a poor competitor (lanes 6–8). To gain a deeper insight into the effect of the 12-nt insertion, deletion mutation towards the HCLV 3 UTR was preformed, as described previously (Xiao et al., 2004a). The HCLV 3 UTR mutant with deletion of the 12-nt insertion was formed, designed as H3 . The 12-nt insertion was introduced into the Shimen 3 UTR, the Shimen 3 UTR mutant was formed, designed as S3 + (Fig. 5). The competitive EMSA towards H3  and S3 + was done. The results showed that the H3  almost had same high binding activity as S3 while S3 + and H3 competed poorly (Fig. 7). These results showed that the 12-nt insertion, CUUUUUUCUUUU, was deleterious to helicase binding.

3.6. Effects of secondary structure of 3 terminal sequences of 3 UTR on helicase binding It has been shown that the secondary structure of 3 UTR is important in NS3 helicase binding (Banerjee and Dasgupta, 2001). To reinforce the finding with our results about CSFV, we analyzed the effects of secondary structure of 3 terminal sequences of 3 UTR on RNA binding of the helicase. Secondary structures of the 3 terminal sequences of 3 UTR mutants were predicted with the software, and compared with that of 3 terminal sequences of wild-type 3 UTR. The plus-strand 3 UTR was first addressed. Deletion of 3 consecutively terminal cytidines and 3 CCCGG changed basic pattern of the first stemloop (SL1) of the secondary structure (Fig. 8, PT15 and 16).

Fig. 7. Effects of a 12-nt insertion present in the 3 UTR on helicase binding. The competitive EMSA was preformed with the NS3H and the 32 P-labeled PT0 in the presence (lanes 3–14) of increasing amount (lanes 3–5, 10, 25 and 50 pmol) of various competitors. Lane 2 represents for free PT0 probe to which no NS3H was added, lane 1 for without competitors.

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Fig. 8. The predicted secondary structures of the plus-strand 3 UTR, the minus-strand 3 UTR and their mutants. The software for prediction of secondary structures of RNA templates is RNAstructure 3.2. Maximum % energy difference, maximum number of structures, and window size are designed as 10, 20 and 5, respectively. The names of templates were indicated on the top. Only that of the first stem-loop (SL1) was shown. The number represents the positions of 3 -most terminal nucleotide. The suspension points (. . .) represent the other structural parts.

Deletion of the 21-nt 3 terminal sequence seriously destroyed the whole pattern of the secondary structure of 3 UTR (results not shown). These deletions seriously reduced the binding of 3 UTR to NS3 helicase (Fig. 6A, lanes 8–16). In the case of the minus-strand 3 UTR, similar results were found. The predicted secondary structure of the wild-type 3 UTR contains an intact stem-loop structure at 3 terminus (Fig. 8, MT0). The stem-loop structure was destroyed in the predicted secondary structure of the 3 UTR mutant with deletion of 21-nt fragment, “CTCGTATAC”, “TCGTATAC”, and “GTATAC” at the 3 -most terminal sequence of minus-strand 3 UTR (results not shown). These mutations have been shown to reduce helicase binding (Fig. 6B, lanes 5–16). An intact stem-loop structure was found to be present in the predicted secondary structure of the 3 UTR mutant with deletion of the first 3 terminal cytidine of minusstrand 3 UTR (Fig. 8, MT1). The 3 UTR mutant still has higher binding activity (Fig. 6B, lanes 2–4). Taken together, the 3 terminal intact stem-loop structure in the secondary structure of plus- and minus-3 UTR might be necessary for the CSFV NS3 helicase binding. 4. Discussion All RNA helicases are divided into three superfamilies (SF1–SF3). The nsP2 protein of alphavirus is classified in SF1; the picornavirus-like (2C-like) proteins are grouped under SF3; the NS3 helicases of HCV and BVDV belong to SF2 (Kadare and Haenni, 1997; La´yn´ et al., 1989). In addition to nucleic acid unwinding activities, these proteins also exhibit hydrolysis activities and NTPase activities (Bartenschlager et al., 1993; Grakoui et al., 1993, 1994; Kamer and Argos, 1984; Jin and

Peterson, 1995; Preugschat et al., 1996; Warrener and Collett, 1995; Xu et al., 1997). These proteins all contain common motifs associated with NTP binding, helicase activities and hydrolysis activities (Gorbalenya et al., 1988; Gorbalenya and Koonin, 1989; Hodgman, 1988). The CSFV NS3 protein also possesses these amino acid motifs, therefore postulated to have the RNA helicase activity. The CSFV NS3 protein associated with the RNA helicase activity has been demonstrated in our present report. Moreover, the RNA helicase activity is enhanced by the protease domain present in the NS3 protein since the helicase activity of the full-length NS3 protein is higher than that of the truncated NS3 protein (Fig. 2). The finding is consistent with that of HCV (Zhang et al., 2005). However, it was also reported that there not was a significant difference in helicase activity between the full-length and truncated NS3 proteins (Gallinari et al., 1998, 1999). The 3 UTR of plus-strand RNA of CSFV genome is believed to be the first entry site for viral replicases for initiation of RNA genome replication. The helicase activity is needed to unwind the secondary structure present in the 3 UTR for the replication. To examine the binding of the NS3 helicase domain to the 3 UTR, the EMSA experiments were done. Our experiments have demonstrated interaction of the NS3 helicase domain with the plus- and minus-strand 3 UTR. Further, it is found that this interaction appears specific, since binding of NS3H to the ratio-labeled CSFV 3 UTR is successfully competed by the nonlabeled homologous 3 UTR but not by other sequences from the viral genome. The specific interactions between RNA helicases and viral RNA fragments have been observed in other virus. The HCV NS3 protein is observed to specifically bind to the plusand minus-3 UTR (Banerjee and Dasgupta, 2001). In poliovirus,

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the 2C protein specifically binds to the 39-terminal sequences of viral negative strand RNA (Banerjee et al., 1997). Recombinant dengue virus helicase also exhibits specific viral RNA binding (Cui et al., 1998). Previous reports show that 3 UTR also is necessary binding site for viral replicase to initiate RNA syntheses (Xiao et al., 2004b). Therefore, the 3 UTR is an important site for interaction between proteins and viral RNA genome. We also observed that the binding activity of the minus-strand 3 UTR is higher than that of the plus-strand 3 UTR in the EMSA experiment with the NS3H. The same results have been obtained in competitive binding experiment with the CSFV NS5B protein, in which the minus-strand 3 UTR is more effective than the plusstrand 3 UTR in NS5B binding (Xiao et al., 2004b). It is tempting to speculate that the minus-strand 3 UTR is a more effective template for synthesis of its complementary strand RNA than the plus-strand 3 UTR. The speculation coincides with the fact that the HCV RNA polymerase replicates in vitro the 3 terminal region of the minus-strand viral RNA more efficiently than the 3 terminal region of the plus RNA (Reigadas et al., 2001). Indeed, it has been observed that much more plus-strand RNAs than minus-strand RNAs are detected when the BVDV is replicating in its native cells (Gong et al., 1996), even in the cells harboring the HCV RNA replicon (Lohmann et al., 1999). Our previous reports show that the 3 terminal sequence of 3 UTR is necessary for RNA synthesis and replicase binding (Xiao et al., 2004b). To exhibit the function of these 3 terminal sequences in NS3 helicase binding, the competitive binding experiments with the NS3H towards these 3 terminal sequences were done. Results show that deletion of the 21-nt and other 3 terminal sequences of plus- and minus-strand 3 UTR destroys, or reduces binding activity, consistent with many previous reports (Banerjee and Dasgupta, 2001). Our previous reports show the 3 UTR mutants with deletion of 3 terminal sequences is unable to be bound to NS5B proteins, even inhibits RNA synthesis by viral replicase (Xiao et al., 2004b). These findings strengthen the postulation that the 3 terminal sequence of 3 UTR is essential to replicase and helicase activities (Banerjee and Dasgupta, 2001; Gu et al., 2000; Zhang et al., 2005). The 3 terminal sequence of 3 UTR might be also the first interactive site between helicase and viral genome. Interestedly, some mutations within the 21-nt sequence of 3 UTR did not significantly reduce binding activity. But, the same mutations were previously reported to completely destroy the template activity for RNA synthesis (Xiao et al., 2004c). The specificity for template for RNA synthesis might be stricter than that for substrate for RNA unwinding. To clear the reason why deletion of 3 terminal sequences of the 3 UTR reduces the binding activity, the secondary structure was predicted. The predicted secondary structures of the mutated 3 UTRs were compared with that of the wild-type 3 UTR. Results show that an intact secondary structure of 3 terminal sequence of 3 UTR is important in helicase binding, which has been demonstrated to be necessary for replicase binding and RNA synthesis (Xiao et al., 2004b). The CSFV HCLV strain was obtained from the CSFV Shimen strain after 480 passages in the rabbit (Wu et al., 2001). An insertion of 12 continuous nucleotides, CTTTTTTCTTTT, is found in the 3 UTR of HCLV genomic cDNA, compared with its

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parental virulent Shimen strain (Bj¨orklund et al., 1998; Wu et al., 2001). In our competitive EMSA for the NS3 helicase binding, it was observed that the HCLV 3 UTR was a poor competitor than Shimen 3 UTR (Fig. 7). Our previous reports have also shown that the 12-nt insertion resulted in reduction in RNA synthesis. Taken together, these findings reinforce the speculation that the 12-nt insertion might be one of the reasons for avirulence (Xiao et al., 2004a). Taken together, it is showed that the 3 UTR, especially the  3 terminal sequence of the 3 UTR, is the site for interaction between NS3 helicase and RNA genome. We previously reported that the 3 terminal sequence of the 3 UTR also is the site for the interaction between NS5B replicase and viral genome. Therefore, it is postulated that the NS3 helicase might have the same binding site in 3 UTR as the NS5B replicase. The same binding site and the different binding time for different proteins lead to sequent replication of viral genome. In fact, many works have been done about the protein–protein interaction in virus. It has been reported, the HCV NS3 enhances NS5B RdRp activity (Gallinari et al., 1999), the RdRp regulates the functions of NS3 during HCV replication (Zhang et al., 2005). The interaction between NS4B and NS5B has been described (Isken et al., 2004). In HCV, NS4A bind tight to the protease domain of NS3 to form NS4A–NS3 protease domain complex for stabilization of the NS3 structure (Kim et al., 1996; Love et al., 1996; Yan et al., 1998). In fact, direct interaction of NS5B with NS3 and NS4A has been reported (Ishido et al., 1998). Acknowledgements This work was supported by National Natural Science Foundation of China (30670445) and by the Science and Technology Foundation of Shanghai Higher Education (05ZZ14). References Banerjee, R., Dasgupta, A., 2001. Specific interaction of hepatitis C virus protease/helicase NS3 with the 3 -terminal sequences of viral positive- and negative-strand RNA. J. Virol. 75, 1708–1721. Banerjee, R., Echeverri, A., Dasgupta, A., 1997. Polio virus-encoded 2C polypeptide specifically binds to the 39-terminal sequences of viral negativestrand RNA. J. Virol. 71, 9570–9578. Bartenschlager, R., Ahlborn-Laake, L., Mous, J., Jacobsen, H., 1993. Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions. J. Virol. 67, 3835–3844. 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. ˇ Bel´ak, S., 1998. Molecular characBj¨orklund, H.V., Stadejek, T., Vilˇcek, S., terization of the 3 noncoding region of classical swine fever virus vaccine strain. Virus Genes 16, 307–312. Cui, T., Sugrue, R.J., Xu, Q., Lee, A.K., Chan, Y.C., Fu, J., 1998. Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein. Virology 246, 409–417. Cuthbert, J.A., 1994. Hepatitis C: progress and problems. Clin. Microbiol. Rev. 7, 505–532. Fletcher, S.P., Jackson, R.J., 2002. Pestivirus internal ribosome entry site (IRES) structure and function: elements in the 5 untranslated region important for IRES function. J. Virol. 76, 5024–5033.

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