Characterization of avian reovirus non structural protein σNS synthesized in Escherichia coli

Virus Research 67 (2000) 1 – 9 www.elsevier.com/locate/virusres

Characterization of avian reovirus non structural protein sNS synthesized in Escherichia coli Hsien Sheng Yin, Long Huw Lee * Department of Veterinary Medicine, National Chung Hsing Uni6ersity, Taichung 403, Taiwan Received 7 September 1999; received in revised form 13 December 1999; accepted 21 December 1999

Abstract The coding region of avian reovirus S1133 genomic segment S4, encoding the non structural protein sNS, was inserted into expression vector pET28a and the protein was expressed in Escherichia coli BL21(DE3) as a fusion protein containing a C-terminal peptide with six tandem histidines (His-tag). The expressed protein (esNS) consistent with the expected molecular size of the avian reovirus protein sNS synthesized in infected cells was readily purified by His-Bind Resin. The esNS was further confirmed to be indistinguishable from viral sNS by immunoblot analysis. The esNS binds 32P-labeled ssRNA probe produced by run-off transcription of clone pGEM-3Zf( + )S4. The binding activity is blocked by heterologous yeast rRNA, but not by homologous avian reovirus dsRNA and heterologous infectious bursal disease virus dsRNA and salmon sperm dsDNA. Therefore, the ssRNA-binding activity of the expressed protein sNS is non sequence-specific, similar to that previously described for viral sNS purified from avian reovirus infected cell extracts. In addition, the recent data also show that the optimal salt (NaCl) concentration and pH for its binding are 100–150 mM and 7.0, respectively, in terms of the UV cross-linking and RNase A treatment of the reaction mixtures prior to the denaturing gel analysis. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Avian reovirus; Non structural protein sNS; ssRNA-binding; Gel shift

1. Introduction The genome and protein composition of avian reovirus (ARV) are generally similar to those mammalian reoviruses (MRV), the prototype of the genus orthoreovirus (Gouvea and Schnitzer, 1982; Wickramasinghe et al., 1993). Both groups

* Corresponding author. Tel.: + 886-4-2860196; fax: + 8864-2851741. E-mail address: [email protected] (L.H. Lee)

have a genome consisting of ten segments of double-stranded RNA (dsRNA) which was encapsidated by a double-shell capsid. However, ARV differs in its lack of hemagglutination activity and its ability to induce cell fusion (Wilcox et al., 1985). Identification of all virus-encoded proteins showed that virus encoded at least ten primary translation products (Varela and Benavente, 1994) which are separated into three size classes: large (l), medium (m) and small (s). Eight of these products are the structural protein and the other two are non structural.

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In contrast to MRV, ARV proteins have been much less characterized than those of their counterparts. In recent studies using monoclonal antibodies against ARV, protein sC has been indicated to be the target for type-specific neutralizing antibodies (Wickramasinghe et al., 1993); while antibodies to protein sB are responsible to group-specific (Shapouri et al., 1996). Both proteins also involved in induction of cell fusion and may play an important role of viral pathogenesis (Ni and Ramig, 1993; Theophilos et al., 1995; Duncan and Sullivan, 1998). It has been shown that sNS of MRV is able to bind singlestranded RNA (ssRNA) (Huismans and Joklik, 1976; Antczak and Joklik, 1992) and may act in the selection and condensation of the ten different viral ssRNA into precursor subviral particles in preparation for dsRNA synthesis. Similarly, ARV sNS, a counterpart of MRV sNS, present in the ARV S1133-infected cell extracts has been shown to bind poly (A)-Sepharose and the homologous or heterologous ssRNA was able to compete for binding of this protein to poly (A)-Sepharose, suggesting that ARVsNS binds to ssRNA in a nucleotide sequence non specific manner (Yin and Lee, 1998). To further insight into the RNA-binding features of ARVsNS, the S4 genome segment of ARV strain S1133 has recently been cloned and sequenced (Chiu and Lee, 1997). Analysis of the sequence indicates that it possesses a conserved pentanucleotide sequence UCAUC at the 3%-terminus of its plus strand like in other known ARV genome segments S1 (Shapouri et al., 1995), S2 (Yin et al., 2000) and S3 (Yin et al., 1997) and ten segments of MRV (Antczak et al., 1982). The sequence contains a long potential open reading frame (ORF) which was assumed to be the gene coding for sNS, since this protein has previously been assigned to the S4 gene and the deduced amino acid sequence of this ORF produced a protein of the size expected for sNS. In recent studies, we cloned the coding region for ARVsNS into an Escherichia coli (E. coli ) expression plasmid. This approach provided a sufficient amount of sNS in a soluble form to allow us to extend in vitro studies on its biological properties.

2. Materials and methods

2.1. Virus and 6iral RNA extraction A commercially available vaccine strain of ARV S1133 (Vineland Laboratories) was used in this study. The virus has been adapted for growth in chicken embryo fibroblasts (CEF) and plaquepurified twice as described previously (Wu et al., 1994). The virus was propagated in CEF and dsRNA was extracted from the purified virions essentially as described (Wu et al., 1994).

2.2. Labeling of ARV-infected cells with [ 35S] -methionine The procedures for labeling of ARV-infected cells with [35S]-methionine were made essentially as described (Yin and Lee, 1998). Briefly, CEF monolayers were infected with ARV S1133 or mock-infected with alone (M199 supplemented with 2% fetal calf serum) and labeled with [35S]methionine (Amersham Life Science; sp. act. \ 1000 Ci/mmol; 100 Ci in 5 ml Dulbecco’s modified Eagle’s medium without methionine). The cells were lysed and cytoplasmic extracts were prepared.

2.3. pET28a-sNS construction and sNS expression Expression of ARV sNS for its characterization, the ORF coded for sNS in S4 genome was amplified and cloned into E. coli. The forward primer (5%-GTATAGGATCCATGGCGCGTGCCATATAC-3%) corresponded to the 5% region of sNS gene and incorporated a BamHI restriction site (underlined) immediately upstream of the initiation codon of the ORF on the S4 genome segment. The reverse primer (5%-GACTATCTCGAGCTAGGCGGTAAAA-GTGG-3%) was complementary to the 3%s end of the sNS gene and contained a XhoI restriction site (underlined) immediately downstream of the termination codon. The procedures for the preparation of the first strand cDNA from ARV genomic RNA for amplification of the NS coding region were essentially the same as described (Chiu and Lee, 1997).

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Briefly, ARV genomic RNA was separated on 6.5% polyacrylamide gel and the S4 RNA was eluted with an RNaid kit (Bio 101). S4 RNA was denatured with 100 mM methylmercuric hydroxide prior to reverse transcription for the first strand cDNA synthesis using the forward primer. The sNS coding region was subsequently amplified by both primers with Tag polymerase. pET28a-sNS was constructed by insertion of the amplified cDNA fragment into pET28a and used to transform E. coli HMS 174 (Yin et al., 1997). pET28a-sNS was expected to express a protein that contained all 367 amino acid residues encoded by the ARV S4 gene and six histidine molecules at the C-terminal end. Expression of sNS was made in E. coli BL21 (DE3) by the induction of 1 mM IPTG essentially as described previously (Yin et al., 1997). To analyze the bacterial cell samples, cell pellets of cultures induced with IPTG for 4 h were resuspended in sample buffer and boiled for 3 min before sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970).

2.4. Purification of the expressed protein sNS To determine the expressed protein s NS (esNS) in soluble or in insoluble fraction, whole cell pellets from the cultures were disrupted by sonication in TN buffer (20 mM Tris – HCl, 100 mM NaCl, pH 7.9) and the homogenates were centrifuged at 100 000×g for 30 min. The pelleted insoluble materials and the supernatant were analyzed by SDS-PAGE. Additionally, the expressed protein in supernatant fraction was further purified using a His-Bind Resin column (Novagen) according to the manufacturer’s instruction manual. Following washing with the TN buffer, proteins were eluted with 3 ml of TN buffer — 60 mM, 100 mM, 200 mM and 1000 mM imidazole, respectively. The nature of the samples eluted was then analyzed by SDS-PAGE and Western blotting.

2.5. Preparation of ssRNA probe A recombinant plasmid pGEM-3Zf( +)S4 was constructed to prepare the 32P-labeled virus -spe-

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cific ssRNA probe by run-off transcription. The forward primer was identical to nucleotides 1141 to 1154 of the S4 mRNA (numbered according to Chiu and Lee, 1997) and incorporated an Eco RI restriction site at 5% end; the reverse primer was complementary to nucleotides 1161 to 1185 containing a HindIII restriction site at its 5% end. Both primers were used to amplify a cDNA fragment by Taq polymerase using the ARV S4 cDNA (Chiu and Lee, 1997) as the templates. The amplified products were digested with Eco RI and HindIII and subsequently ligated into the corresponding sites of the pGEM-3Zf( + ) in vitro transcription vector (Invetrogen). The constructed plasmid, named as pGEM-3Zf(+)S4 was used to transform E. coli DH5a and plasmid DNA from ampicillin-resistant bacterial was isolated. The expected nucleotide sequence of pGEM-3Zf(+ )S4 was confirmed by dideoxynucleotide sequencing (Sanger et al., 1977). The 32P-labeled virus-specific ssRNA probe was prepared by using T7 RNA polymerase to produce a 56-nucleotide RNA transcript from Eco RI-linearized pGEM-3Zf(+ )S4 according to the manufacturer’s instruction (Invetrogen). At the end of reaction, the template DNA was removed by treatment with RNase-free DNase I. Run-off products labeled with [a-32P] CTP (300 Ci/mmol, Amersham) were gel-purified by electrophoresis on 6% polyacrylamide gel as described previously (Konarska and Sharp, 1987) and used for binding reactions with the purified esNS.

2.6. Gel shift assay Purified e a NS and 32P-labeled ssRNA probe (5×104 cpm) were added to the binding buffer (150 mM NaCl, 1 mM DTT, 0.5% Tween 20, 20 mM Hepes, 5 mM MgOAC, 10% glycerol, pH 7.4) for a total reaction volume of 15 ml and the binding reaction was carried out at 37°C for 15 min. The reaction mixtures were then separated by electrophoresis on 6% polyacrylamide non denaturing gels (Konarska and Sharp, 1987). Gels were dried and subjected to autoradiography for 18–24 h. To dermine pH and ionic strength optima for RNA binding, the desired pH or the molarity of

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NaCl was adjusted as indicated. Purified esNS (6 mg) was incubated with 32P-labeled ssRNA probe (5× 104 cpm) under modified conditions at 37°C for 15 min. After cross-linking of RNA to purified esNS with UV light, the mixtures were treated with RNase A (see below) and then were separated by electrophoresis on 6% denaturing polyacrylamide gels (Laemmli, 1970) and visualized by autoradiography. Blocking of the purified esNS binding activity was analyzed by amending the above binding reactions with 1000 ng of heterologous RNA (yeast rRNA), homologous avian reovirus dsRNA and heterologous infectious bursal disease virus dsRNA (Lee et al., 1994) and salmon sperm DNA. After incubation at 37°C for 15 min 32P-labeled ssRNA probe (5 × 104 cpm) was added to the mixture and incubated at 37°C for additional 15 min.

2.7. Cross-linking of RNA to the esNS with u6 light The reaction mixtures containing the purified esNS and ssRNA probe were placed on ice and exposed to UV light for 10 min using a 254 nm 25 W germicidal lamp placed 3 – 4 cm above the samples. For immunoprecipitation, following exposure a half volume of samples was treated with 0.5 mg/ml of RNase A at 37°C for 10 min and extracted with phenol/chloroform twice. The same conditions were used to treat the samples to determine pH and ionic strength optima for RNA binding.

2.8. Immunoprecipitation Immunoprecipitation was used to recover esNS from the UV cross-linked, RNase A digested samples as follows. The samples (7.5 ml) were mixed with binding buffer to a total volume of 500 ml. Anti-sNS antiserum was added to a final dilution of 1:50 and the samples were incubated for 1 h at 25°C. Protein G Agarose (Bohringer Mannheim) (10 ml) was added and incubated for additional 1 h at 25°C with gentle rotation. After centrifugation, the beads were washed three times in a buffer containing 100 mM NaH2PO4, 100 mM

NaCl, 0.1% SDS, 0.1% Triton X-100, 5 mM deoxycholate, pH 7.4). Proteins were eluted from the beads by boiling in sample buffer and analyzed by SDS-PAGE and autoradiography. The same conditions were used to immunoprecipitate proteins presence in the virus-infected cytoplasmic extracts as prepared above.

2.9. Preparation of antiserum and Western blotting Monospecific antibody against purified viral sNS (Yin and Lee, 1998) was prepared in BALB/ c mice. The animals were intraperitoneally injected with the purified viral sNS (35 mg per mouse) emulsified in complete Freund adjuvant. Two additional boosts with the same amount of proteins in incomplete Freund adjuvant were given every other week. Sera were collected at 2 weeks after final boost. Western blotting was then performed by using a 1:350 dilution of the above antisera to identify the expressed viral proteins esNS as described previously (Yin et al., 1997).

3. Results

3.1. Expression and purification of esNS After induction of E. coli containing the pET28a-sNS construct with IPTG, both cell extracts and insoluble pellets were analyzed by SDSPAGE. The results indicated that a protein of : 40.6 kDa accumulated to high levels was present in both fractions (Fig. 1A, lane 4) and was consistent with the expected molecular size of the ARV protein sNS (Huismans and Joklik, 1976; Wickramasinghe et al., 1993; Varela and Benavente, 1994). This protein in cell extracts was then purified using His-Bind Resin. Proteins in a fraction eluted with TN buffer — 100 mM imidazole produced a single band, which was homogenous (Fig. 1B, lane 3). The nature of the purified, expressed protein was further verified by using a monospecific antiserum to the purified ARVsNS. Western blot assay showed that the expressed protein, despite it was purified or not, reacted specifically with the antiserum (Fig. 1C, lanes 2

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and 3), although some other minor bands in non purified sample were also present (Fig. 1C, lane 2). These bands were probably generated as a result of the degradation of the expressed protein as they were not evident in the extracts of cells bearing the parent pET plasmid (Fig. 1C, lane 1). A visible band with high molecular weight was also recognized. This could be due to overloading of the samples in which protein molecules were not well-dissociated after boiling. The results suggest that the expressed protein, esNS, is encoded by the sNS coding region of the ARV S4 gene and is virtually indistinguishable from the ARVsNS.

3.2. Detection of RNA-binding of esNS by gel shift analysis

Fig. 1. Expression, purificationand identification of protein esNS. (A) sNS was expressed in E. coli BL21 (DE3) as a fusion protein. Whole cells were sonicated and centrifuged. The pelleted materials (P) and supernatants (S) were separated on SDS-PAGE, respectively and stained with Coomassie blue. Lanes 1 (BL21, no pET28a), 2 (BL21 with pET28a but no insert)and 4 (clone pET28a-sNS) was the samples in which expression was induced with IPTG after a further 4 h, starting at 2 h after the OD600 of 0.6 was obtained. Lane 3 (clone pET 28a-sNS) was the samples collected immediately after the addition of IPTG. Protein molecular weights in kDa are presented on the left; (B) The sample lanes 1, the supernatant of bacterial cells with pET28a but no insert; 2, the supernatant prepared as indicated for (A); lane 3, the sample purified from the supernatant prepared as indicated for lane 2 by using His-Bind Resin. Protein molecular weights are as indicated for (A). All samples were analyzed on SDS-PAGE followed by staining with Coomassie blue. (C) Immunoblot with a mouse anti-purified ARVsNS, 1:350 dilution was performed for analysis of the reactively specific to esNS. Lanes 1 to 3 were the samples as for (B).

To test nucleic acid-binding activity of esNS, binding assay was made by incubating esNS with the labeled ssRNA probes. Fig. 2A showed that the incubation resulted in the formation of RNA-protein complexes that were separated from the free RNA probe during native gel electrophoresis. The progressive slowing of the mobility of the complexes as the esNS concentration was increased suggested that multiple esNS molecules were binding to the RNA probe. As an alternate mean toward the identification of the esNS responsible for gel shift, the reaction mixtures were exposed to the uv light to induce covalent cross-linking of the RNA probe to any intimately associated proteins (Smith, 1976). To facilitate the estimation of protein molecular weight, a portion of the crosslinked materials was digested with RNase A removing all but the few nucleotides of the RNA probe which were directly linked to the protein. Then, immunoprecipitation with the anti-sNS antibody was used to recover esNS from esNSRNA probe mixtures which had been crosslinked and RNase A digested. The UV cross-linked mixtures and immuonprecipitates were then analyzed for the presence of 32P-labeled proteins by SDS-PAGE followed by autoradiography. Upon RNase A untreatment of the cross-linked mixtures, it failed to reveal the

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Fig. 2. Analysis of esNS binding to ssRNA. (A) Binding reaction (as described in Section 2) with 32P-labeled ssRNA probe (5 ×104 cpm) and esNS at the concentrations of 0 (lane 1), 5 (lane 2), 40 (lane 3), 200 (lane 4), 4000 (lane 5) and 8000 (lane 6) ng. The reaction mixtures were electrophoresed through the native polyacrylamide gels and autoradiographed. (B) Cross-linking of RNA to the purified esNS with UV light. Six micrograms of the purified esNS and 32P-labeled ssRNA probe (5 ×104 cpm) were mixed and exposed to UV light as described in Section 2. After exposure, the samples were directly analyzed by SDS-PAGE (lane 2) or the samples were subsequently treated with RNase A followed by immunoprecipitation of 32P-labeled proteins with anti-sNS antiserum and analyzed on the same gel (lane 3). The ARVsNS presence in the cytoplasmic extracts prepared from mock-infected (lane 4) or ARV-infected (lane 5) CEF labeled with [35S] methionine was immunoprecipitated with the same antiserum. The positions of the 32 P-labeled protein ( ’ ) and 35S-labeled sNS (filled arrow head) are indicated. Lane 1 contains ssRNA probe without esNS.

discrete protein bands shifted (Fig. 2B, lane 2). Analysis of the precipitates showed that anti-sNS precipitated a radiolabeled protein (Fig. 2B, lane3) which was slightly greater than 35S-labeled protein sNS precipitated from the virus-infected cell extracts using the same antibody (Fig. 2B, lane 5). This result indicated that the protein to which a few RNase-resistant nucleotides were cross-linked and were transferred was indeed esNS. In addition, the gel shift activity was not noted with reactions in which the purified esNS had been heat treated or the reaction was amended with detergent or proteinase K (Fig. 3). These results indicated that the shift of RNA-protein complexes in the gel is due to an interaction of RNA probe with esNS. The addition of bovine serum albumin (BSA) was used to assess whether non specific protein interactions could be the cause of shift (Fig. 3, lane 6). As no shift was noted with this substitution, binding does not appear to be due to non specific protein-nucleic acid interaction.

3.3. Sequence specificity of the RNA-binding of esNS Binding of esNS to ssRNA was sequence independent since RNA-protein formation was not observed when heterologous yeast rRNA was used as a competitor (Fig. 4, lane 4). Homologous ARV dsRNA (Fig. 4, lane 3) and heterologous infectious bursal disease virus dsRNA (Fig. 4, lane 5) and salmon sperm DNA (Fig. 4, lane 6) were not able to compete as the gel shift was seen, indicating that esNS did not bind to dsRNA and DNA.

3.4. Effects of ionic concentration and pH on the binding acti6ity of esNS To avoid the binding reaction of esNS and RNA probe under different ionic concentrations and pHs from the effects of running buffer during non denaturing gel electrophoresis, the reaction mixtures were UV-linked and were directly assayed by SDS-PAGE and autoradiography. This

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analysis failed to provide a clear result for the evaluation of the differences among different reaction conditions as the RNA-protein complexes appeared as an ill-defined smear (data not shown). The ability of esNS to interact with ssRNA under different conditions was thus determined by UV cross-linking of the reaction mixtures followed by RNase A treatment before gel shift analysis with SDS-PAGE. Fig. 5 showed that the binding of esNS to ssRNA was not detected at 200 mM NaCl or greater and the optimal salt concentration for binding was 100 – 150 mM. The optimal pH for ssRNA binding of esNS was tested. The binding was detected over a wide range with an apparent optimum at pH 7.0.

Fig. 4. Autoradiograph of nucleic acid binding specificity of esNS after electrophoresis through a native polyacrylamide gel. Binding reactions containing esNS (6 g) and 32P-labeled ssRNA probe (5 ×104 cpm) (lane 2) were amended with 1000 ng of unlabeled heterologous competitor yeast rRNA (lane 4) and homologous ARV dsRNA (lane 3) and heterologous infectious bursal disease virus dsRNA (lane 5) and salmon sperm dsDNA (lane 6) prior to incubation. Lane 1, ssRNA probe without esNS.

4. Discussion

Fig. 3. Gel shift analysis of esNS binding activity amending with different condition. Binding reactions with 32P-labeled ssRNA probe (5 ×104 cpm) and esNS (6 mg) (lane 2) were either amended with proteinase K (1mg) (lane 3), 0.1% SDS (lane 4), or the esNS was heated to 100°C for 5 min prior to binding reactions (lane 5). The reaction mixtures were analyzed through the native polyacrylamide gel and autoradiographed. Lane 1, 32P-labeled ssRNA probe. Lane 6, BSA (6 g) with 32P-labeled ssRNA probe.

In this study, we have constructed an expression plasmid that synthesized the ARVsNS in E. coli and extended the findings of Yin and Lee (1998) by using gel shift analysis. The expressed protein esNS was identified as the protein sNS encoded by the coding region of ARV S4 genome segment by immunoblot analysis with antibody raised against the purified ARV sNS from the infected CEF cells. In addition, several nucleic acid-binding properties of esNS described here were similar to those reported for the purified viral protein sNS (Yin and Lee, 1998), in particular, the affinity of sNS for ssRNAs and no detectable of binding to dsRNA and dsDNA. The esNS showed no preference for ARV-specific ssRNA over other heterologous ssRNA (Yeast rRNA) because yeast rRNA competed the bind-

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ing of esNS to virus-specific ssRNA probe. Viral sNS showed a similar lack of preference, also binding heterologous ssRNA (Yin and Lee, 1998). Thus, esNS bound specifically to ssRNA in a sequence-independent manner, of which has been characterized as a common feature for several ssRNA binding proteins (Antczak and Joklik, 1992; Zhao et al., 1994; Patton, 1995). The similarities between esNS and viral protein sNS thus suggest that the esNS synthesized in E. coli is virtually undistinguishable from that produced in infected CEF cells. The binding activity of MRV sNS for ssRNAs has suggested that its primary function during viral replication is to bind ssRNAs, presumably reovirus mRNAs. A previous work suggested that MRV sNS was involved in the selection and condensation of ten different reovirus mRNAs (Huismans and Joklik, 1976). Thus, sNS would be expected to have greater specificity for reovirus mRNAs. However, this is not the case for either purified viral sNS or the purified esNS that both bind a variety of heterologous ssRNAs (Huismans and Joklik, 1976; Richardson and Furuichi, 1985). Recently, it has been shown that components of assorting particles could be precipitated from in-

fected cells using monoclonal antibodies directed against MRV proteins. Their results suggested that sNS, as well as mNS and s3, associated with individual mRNA molecule to form nucleoprotein complexes soon after transcription and the genome segment assortment into progeny genomes and minus-strand synthesis may be intimately linked (Antczak and Joklik, 1992). Our previous work (Yin and Lee, 1998) and the recent data revealed that the properties of the binding activities of either purified esNS or purified ARV sNS for ssRNA are very similar to those of MRV. Functionally, ARV sNS may play a similar role in the early stage of its transcription and replication as described for mammalian reovirus. The N-terminus of the MRVsNS (the first 11 amino acids) has previously been shown to be one of the more highly conserved regions among the MRVsNS proteins of three serotypes (Wiener and Joklik, 1987). Recently, this region, which is predicated to form an amphipathic a-helix, has been indicated to be important for its ssRNAbinding (Gillian and Nibert, 1998). However, this amphipathic-helical nature at the N-terminal sequence in the MRVsNS has been shown to be not conserved in several fusogenic orthoreovirus

Fig. 5. Autoradiograph of the effect of salt concentrations and pH on nucleic acid binding by esNS after cross-linking and RNase-treated and analyzed by SDS-PAGE. The esNS (6 mg) by 32P-labeled probe (5 ×105 cpm) binding reactions were modified to have the indicated pHs and NaCl concentrations. The 32P-labeled RNA probe esNS protein complexes (filled arrow head) are indicated.

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sNS, including ARV, suggesting that an amphipathic helix at the N-terminus of sNS may not be required for ssRNA binding (Duncan, 1999). The ARVsNS has been reported as a ssRNAbinding protein (Yin and Lee, 1998). To illustrate the ssRNA-binding domain of the ARVsNS, the deleted mutant proteins of the ARVsNS are currently being prepared for further studies.

Acknowledgements This research work was supported by the National Science Council (NSC86-2321-B005-051), Republic of China.

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