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1 Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan
2 Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 912, Taiwan
3 Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing Hua University, Hsinchu 300, Taiwan
Correspondence
Long Huw Lee
lhlee{at}mail.nchu.edu.tw
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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phosphoanhydride bond of all four NTPs, since NDPs were the only radiolabelled products observed. The substrate preference was ATP>CTP>GTP>UTP, based on the estimated kcat values. Alanine substitutions for lysines 408 and 412 (K408A/K412A) in a putative nucleotide-binding site of µA abolished NTPase activity, further suggesting that NTPase activity is attributable to protein µA. The activity of µA is dependent on the divalent cations Mg2+ or Mn2+, but not Ca2+ or Zn2+. Optimal NTPase activity of µA was achieved between pH 5.5 and 6.0. In addition, µA enzymic activity increased with temperature up to 40 °C and was almost totally inhibited at temperatures higher than 55 °C. Tests of phosphate release from RNA substrates with µA or K408A/K412A µA indicated that µA, but not K408A/K412A µA, displayed RTPase activity. The results suggested that both NTPase and RTPase activities of µA might be carried out at the same active site, and that protein µA could play important roles during viral RNA synthesis. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). At least 13 primary translation products are encoded by the virus genome. Eight of them are structural components of the viral particle and the other five are non-structural proteins (Bodelon et al., 2001; Shmulevitz et al., 2002; Varela & Benavente, 1994). Protein 
A, encoded by the S2 gene of avian reovirus, possesses dsRNA-binding activity (Yin et al., 2002) and is involved in the resistance of avian reovirus to interferon (Martinez-Costas et al., 2000).
Mammalian reoviral proteins µ2 (Kim et al., 2004; Noble & Nibert, 1997b) and 
1 (Noble & Nibert, 1997a; Bisaillon et al., 1997; Bisaillon & Lemay, 1997) and rotavirus protein NSP2 (Vasquez-Del Carpio et al., 2006) have been reported to be associated with nucleotide triphosphatase (NTPase) and RNA triphosphatase (RTPase) activities. To identify which avian reoviral proteins are responsible for NTPase or RTPase activity, our previous work indicated that avian reovirus protein A possessed NTPase activity (Yin et al., 2002). In the present report, µA has been synthesized in an Escherichia coli expression system. After purification and identification, both NTPase and RTPase activities of the recombinant protein µA ( µA) have been characterized. The results show that µA contains the active site of NTPase activity, which enables the protein to hydrolyse each of four NTPs to their corresponding nucleotide diphosphates (NDPs). The substrate preference was ATP>CTP>GTP>UTP, based on the estimated kcat values. Alanine substitutions for lysines 408 and 412 (K408A/K412A) in a putative nucleotide-binding motif of µA abolished its NTPase activity, further suggesting that NTPase activity is attributable to protein µA. Phosphate-release assays from RNA substrate with µA or K408A/K412A µA indicated that µA, but not K408A/K412A µA, displayed RTPase activity. The results suggested that both NTPase and RTPase activities might be carried out at the same active site and that protein µA could play important roles during viral RNA synthesis.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Viruses were propagated in CEF cells for 36–48 h. Virions were purified and viral dsRNA was released by proteinase K digestion in the presence of 0.1 % SDS (Yin et al., 1997), and further isolated by LiCl fractionation and precipitation (Diaz-Ruiz & Kaper, 1978).
Preparation of viral cores.
Viral cores were generated as described previously (Schnitzer et al., 1982; Su et al., 2006). Briefly, purified reovirions were digested in 100 µl chymotrypsin reaction buffer (0.05 M NaCl, 0.015 M sodium citrate, pH 8.0), containing 200 µg chymotrypsin ml–1 (Sigma-Aldrich), at 37 °C for 60 min. Reactions were terminated by adding 2 µl PMSF (100 mM). Digested particles were pelleted and were analysed by SDS-PAGE (Laemmli, 1970) to confirm the digestion of µB/µBC to form 
and the removal of B (Duncan, 1996; Su et al., 2006).
Construction of vector for expression of µA protein.
Purified viral dsRNA was used to generate a cDNA corresponding to the entire open reading frame (ORF) encoding µA, in the M1 segment, by RT-PCR (Yin et al., 2000). The sequences and locations of primers were chosen according to the cDNA sequence of the avian reovirus S1133 M1 segment (Su et al., 2006). The forward primer (5'-TCCCCGAATTCATGGCCTATCTAGC-3') corresponds to the 5' region of the gene encoding µA (nt 13–26), and incorporates an EcoRI restriction site immediately upstream of the initiation codon of the ORF on the M1 segment (underlined). The reverse primer (5'-CCGCTCGAGTCAGTGCTCGCCTCC-3') is complementary to the 3' end of the gene encoding µA (nt 2211–2197), and contains an XhoI restriction site immediately downstream of the termination codon (underlined). Both primers were used to amplify the µA-encoding region with Taq polymerase (Protech technology). The amplified products were cloned into TOPO II TA cloning vector (Invitrogen). After sequencing, the cDNA of µA was subcloned into the pET28a expression vector (Novagen) to generate pET28a µA (Yin et al., 2000). pET28a µA was expected to synthesize an 84.8 kDa recombinant protein, µA, which includes all 732 aa encoded by the µA-encoding region, a 4.5 kDa peptide and six histidine residues at the N-terminal end.
Construction of vector for expression of mutant protein K408A/K412A µA.
It has been shown that alanine substitutions for lysine 415 and 419 (K415A/K419A) in a nucleotide-binding region of mammalian reovirus µ2 specifically abolished its NTPase and RTPase activities (Kim et al., 2004). To further determine whether point mutations within the region of µA that was predicted to be involved in NTPase activity might eliminate this enzymic activity, point mutants of the µA-encoding region were generated using two PCR and four primers. Briefly, pET28a µA was used as the template and mutations at amino acid residues 408 and 412 were generated by PCR using a 5' sense primer, 5'-TCCCCGAATTCATGGCCTATCTAGC-3', corresponding to nt 13–26 and incorporating an EcoRI site immediately upstream the primer (underlined), and an internal antisense primer, 5'-TCCAATGGAATCTATCCACC-3', corresponding to nt 1209–1190; or an internal sense primer, 5'-CTGTGTATGCCTGCAGGTTCTTTCGCGTCAACTATGATTAAATTTC-3', corresponding to nt 1210–1255, and a 3'-antisense primer, 5'-CCGCTCGAGTCAGTGCTCGCCTCC-3', corresponding to nt 2211—2197, incorporating an XhoI site (underlined). The amplified DNA products were gel-purified and combined in blunt-end ligation buffer in the presence of E. coli T4 DNA ligase. The full-length mutant DNA, bearing sequence changes at both aa 408 and 412, was gel-eluted and used to generate recombinant plasmid pET28a-K408A/K412A µA for K408A/K412A µA protein expression, as described for µA expression.
Expression, purification and identification of µA and K408A/K412A µA.
To express µA and K408A/K412A µA for their functional assay, either µA or K408A/K412A µA was synthesized in E. coli BL21(DE3) and purified as described previously (Yin et al., 2000). Briefly, pET28a µA or pET28a-K408A/K412A µA construct was used to transform E. coli BL21 (DE3) and protein synthesis was carried out by induction with 1 mM IPTG for 4 h. To obtain soluble or insoluble expressed proteins, whole bacterial cell pellets were disrupted in a binding buffer (500 mM NaCl, 20 mM Tris/HCl, pH 7.9) by sonication at 4 °C, and the homogenates were centrifuged at 128 000 g</italic> for 30 min. The pelleted material and the supernatant were assessed on polyacrylamide gels (Laemmli, 1970). Proteins in the supernatant were further purified using a His-Bind resin column (Novagen) according to the manufacturer’s instructions. After washing with the binding buffer, the expressed proteins were eluted with binding buffer containing variable concentrations of imidazole. The nature of the samples eluted was then analysed by SDS-PAGE and Western blotting using an anti-His monoclonal antibody (Amersham Biosciences) or polyclonal antiserum raised against avian reovirus as the probe. To further confirm that the expressed µA or K408A/K412A µA is encoded by the µA- or K408A/K412A µA-encoding region of the M1 segment, respectively, the gels containing bands with the expected size after SDS-PAGE were cut and removed for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis (Voyager-DE PRO; Applied Biosystems). Peptide masses obtained were searched against a comprehensive, non-redundant protein sequence database (NCBI nr) using the Mascot program (Perkins et al., 1999) for protein identification. To assess whether the mutation resulted in structural change of K408A/K412A µA, purified K408A/K412A µA from soluble fractions was directly treated with V8 protease (Sigma-Aldrich) in the stacking gel during subsequent electrophoresis (Yin et al., 1997). Patterns of peptide fragments were then visualized after Coomassie blue staining.
Antiserum preparation and Western blotting.
Antiserum against avian reovirus S1133 was prepared in BALB/c mice. The animals were injected intraperitoneally with the purified virions emulsified in complete Freund's adjuvant. Two subsequent boosts with the same amount of virions in incomplete Freund's adjuvant were given every 2 weeks. Sera were collected at 10 days after final injection. A 1 : 2500 dilution of the above antiserum or an anti-His monoclonal antibody (Amersham Biosciences) was used as the probe in Western blotting (Yin & Lee, 2000).
NTPase reactions and colorimetric assay for phosphate ion.
To estimate the Vmax, KM and kcat values of µA in hydrolysis of each NTP, the NTPase reactions were carried out in 1.5 ml microcentrifuge tubes and then transferred onto a 96-well microtitre plate for development and measurement of phosphate (Noble & Nibert, 1997a). Reaction mixtures contained 50 mM HEPES (pH 7.5), 50 mM NaCl, 5 mM MgCl2, variable concentrations of each NTP and 1.2 µg µA or K408A/K408A µA in a total volume of 50 µl. After incubation at 37 °C for 10 min, the reaction was terminated with an equal volume of 10 % trichloroacetic acid (TCA). For the measurement of the amount of phosphate in each sample, 100 µl of each reaction mixture was transferred to a 96-well plate and mixed with an equal volume of colorimetric reagent (1 vol. 3 M sulphuric acid, 3 vols 0.8 % ammonium molybdate and 1 vol. 10 % ascorbic acid). The mixture was incubated at 37 °C for 30 min and A655 was measured with a µQuant spectrophotometer (Bio-Tek Instruments). In each experiment, samples containing each of four NTPs, but no protein, were included as controls to estimate background phosphate release due to non-enzymic hydrolysis of NTPs. A standard curve generated with a dilution series of KH2PO4 was used to convert the A655 values to amounts of inorganic phosphate (Pi) released per reaction [mean absorbance value of 0.025±0.003 (nmol phosphate ion)–1].
Thin-layer chromatography (TLC) assay for NTP hydrolysis products.
NTP hydrolysis products of µA were examined by TLC as described previously (Taraporewala et al., 1999; Yin et al., 2002). The reaction mixtures contained 50 mM HEPES (pH 7.5), 50 mM NaCl, 5 mM MgCl2 and 0.5 µCi (18.5 kBq) 
-32P-labelled ATP, GTP, CTP or UTP [specific activity >3000 Ci mmol–1 (111 TBq mmol–1); NEN] and the indicated amounts of proteins or viral cores in a total volume of 10 µl, and were incubated at 37 °C for 30 min. Each reaction mixture (0.5 µl) was spotted onto plastic-backed polyethyleneimine–cellulose TLC sheets (Merck), and the NTP hydrolysis products were separated by ascending chromatography in a solvent containing 1 M formic acid and 0.5 M LiCl or, in some cases, a solvent containing 0.75 M KH2PO4. The TLC sheets were dried and the radiolabelled spots on the sheet were detected by autoradiography. Radiolabelled NTPs treated with 5 U tobacco acid pyrophosphatase (Epicentre) were added as NDP and NMP control markers at the same time.
The properties of the NTPase activity of µA were characterized using [-32P]ATP as previously described (Yin et al., 2002). To determine whether divalent cations are required for the NTPase activity of µA, Ca2+, Mn2+, Mg2+ or Zn2+ was added to the reaction mixtures and was adjusted to yield the desired concentrations as indicated. To determine the optimum temperature for the NTPase activity of µA, reaction mixtures were prepared in a cold room and put on ice, incubated at the appropriate temperature for 30 min, returned to ice and terminated by addition of an equal volume of 10 % TCA. To determine the effects of reaction buffer at different pH values on the NTPase activity of µA, the reaction mixtures were buffered with 50 mM HEPES solution to obtain buffers that spanned a wide range of pH values, from 4.5 to 10.0, and then incubated at 37 °C for 30 min. The percentage activity was calculated from the amount of ATP hydrolysis as measured with a BAS-2500 phosphorimager (Fuji Photo Film).
RTPase activity assay.
A recombinant plasmid, pGEM-3Zf(+)-S4 (Yin & Lee, 2000), was used to generate [-32P]GTP- or [-32P]GTP-labelled 56-mer RNA substrates which contained 11 nt of the vector and 45 nt of avian reovirus S4 segment by run-off transcription as described previously (Yin & Lee, 2000). After treatment by RNase-free DNase I at the end of the reaction, radiolabelled run-off products were gel-purified through a 6 % polyacrylamide gel and used for RTPase activity assay.
Reaction conditions for the RTPase activity assay were identical to the radiographic NTPase assay, except that the indicated amounts of 32P-labelled RNA substrates were used instead of NTPs. Reaction mixtures were either resolved on a 6 % polyacrylamide gel containing 8 M urea and the reaction products were then visualized by exposure to X-ray film, or they were separated by TLC as described for the radiographic NTPase assay.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, lane 2), but it was very faint in soluble fractions (Fig. 1a, lane 3). This protein was consistent with the molecular size expected of the protein µA. Soluble fractions were used for further purification of µA using a His-Bind resin column (Novagen). The eluted protein was stained by Coomassie brilliant blue. The results indicated that µA, eluted from a binding buffer containing 250 mM imidazole after washing with a binding buffer containing 125 mM imidazole, showed a predominant band with the predicted molecular mass of 84.8 kDa (Fig. 1b, lane 4). This band co-migrated with the µA protein from avian reoviral cores during electrophoresis (Fig. 1c, lane 2) and was also recognized by the antiserum against avian reovirus in Western blotting (Fig. 1c, lane 2). The nature of µA was further verified by MALDI-TOF/MS analysis. The results indicated that composition and sequence of amino acids of the purified µA were identified to be the same as those of the protein µA of avian reovirus S1133 (Su et al., 2006). Taken together, these results showed that the purified µA was indeed encoded by the µA-encoding region of avian reovirus M1 gene.
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B were digested; µA was recognized by µA antiserum (Fig. 1c
, lane 1). When viral cores were incubated with each of the four [-32P]NTPs and the products were subsequently resolved by TLC assay, all four NTPs were hydrolysed to their respective radiolabelled NDP and NMP products (Fig. 2, lane 5), suggesting that avian reoviral cores possess NTPase activity.
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-32P]ATP. The products were subsequently resolved by TLC. Fig. 2(a) shows that µA generated ADP as the only radiolabelled product (Fig. 2a, lane 3). The data indicate that the µA protein possesses ATPase activity that enables the protein to hydrolyse the – phosphoanhydride bonds of ATP. ATPase activity was abolished in reactions in which µA had been heat-treated (100 °C) or when the reactions were amended with SDS or proteinase K (Fig. 2b). Protein samples prepared from bacterial cells containing pET28a alone showed that ATPase activity remained at background level (Fig. 2c). Similar results were observed with reactions in which K408A/K412A µA was used (see below). These results indicate that ATPase activity comes from an interaction of µA with ATP. To further characterize whether µA was a non-specific NTPase, 0.4 µg µA was incubated with [-32P]UTP, [-32P]GTP or [-32P]CTP, following the same procedures as described for [-32P]ATP. The results from TLC analysis showed that µA was also able to hydrolyse UTP and GTP, but at a lower level than that obtained from both ATP and CTP hydrolysis (Fig. 2a), suggesting that µA is a non-specific NTPase and catalyses UTP and GTP in a less efficient manner.
Experiments to further characterize the enzymic activity of µA in hydrolysis of each NTP were carried out with 1 µg µA protein in the presence of increasing concentrations of ATP, GTP, CTP or UTP (Fig. 3). Vmax and KM were calculated and the results were presented as a Lineweaver–Burk double-reciprocal plot. As expected, each of four NTPase activities of µA followed Michaelis–Menten kinetics, yielding a straight line on a Lineweaver–Burk plot (data not shown). The results confirmed that µA exhibits a strong preference for ATP and CTP, which were catalysed very efficiently. The protein displayed relatively less activity on both UTP and GTP; the order of preference was ATP>CTP>GTP>UTP, based on the kcat values obtained with these substrates (Table 1).
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). These nucleotide-binding motifs are conserved among avian reoviruses (Su et al., 2006), mammalian reoviruses (Wiener et al., 1989; Yin et al., 2004; Zou & Brown, 1992) and aquareovirus (Attoui et al., 2002), and are very similar to that of alphavirus NSP2 (Rikkonen et al., 1994; Vasiljeva et al., 2000). Therefore, two lysines (K) were substituted by two alanines (A) at positions 408 and 412 in the putative A motif of µA (Fig. 4) to eliminate the apparent NTPase activity of µA, as described for mammalian reovirus protein µ2 (Kim et al., 2004). The mutant protein K408A/K412A µA was expressed in E. coli and purified using the same procedures as those for µA. K408A/K412A µA could be expressed and purified from soluble fractions with the same features as protein µA. The mutant protein co-migrated with µA (Fig. 1b) and core µA (Fig. 1c, lane 3) on SDS-PAGE, and was recognized by antiserum raised against avian reovirus (Fig. 1c, lane 3). Furthermore, the pattern of peptide fragments generated by V8 protease digestion of K408A/K412A µA was similar to that of µA (data not shown), suggesting that the structure of K408A/K412A µA was not significantly altered.
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, lane 4). These results suggest that the putative nucleotide-binding motif of protein µA is involved in NTPase activity.
Characteristics of NTPase activity by µA
The requirement of divalent cations for µA NTPase activity was examined by the addition of Mg2+, Mn2+, Ca2+ or Zn2+ ions into reaction mixtures. The hydrolysis products were again resolved by TLC, detected by autoradiography and quantified using a phosphorimager. The results are indicated in Fig. 5. ATPase activity rapidly increased when the Mg2+ concentration was at 1 mM, and then decreased gradually in the presence of higher Mg2+ concentrations (Fig. 5a). Similar results were observed for the ATPase activity in the presence of Mn2+ (Fig. 5b). However, the ATPase activity of µA remained high, above 50 % of maximum, in the presence of Mg2+ at 1 mM or greater. In contrast, addition of Ca2+ or Zn2+ ion immediately inhibited ATPase activity of µA when the Ca2+ or Zn2+ ion concentration was 1–2 mM, and no significant change was then observed throughout the concentration range of the experiment (Fig. 5c, d). The results suggested that the ATPase activity of µA was dependent on the divalent cations Mg2+ or Mn2+, which could not be substituted by Ca2+ or Zn2+ ion since they inhibited the activity.
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). When the tests were performed using reaction mixtures buffered at different pH, the results showed that ATPase activity rapidly increased with increasing pH values; the optimum activity of µA was achieved between pH 5.5 and 6.0 and then declined in the presence of higher pH values (Fig. 5e).
RTPase activity of µA
To determine whether µA possessed RTPase activity, µA was tested for its capacity to remove the phosphate from ssRNA substrates. This assay indicated that loss of radiolabel from the RNA substrates was observed with the -32P-labelled but not the -32P-labelled GTP substrates (Fig. 6a). The results reveal that µA displays RTPase activity. This activity was further assessed by TLC. The results showed that the amounts of 32P-labelled phosphate released by µA were altered as a function of the concentrations of µA (Fig. 6b). The addition of EDTA abolished the phosphate release from the RNA substrates by µA (Fig. 6, lane 7), indicating that RTPase activity, like NTPase activity, is dependent on divalent cations. The mutant protein K408A/K412A µA did not display RTPase activity in either acrylamide gels or TLC (Fig. 6, lanes 8 and 9), suggesting that RTPase activity might involve the same putative nucleotide-binding region of µA as NTPase activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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1 and µ2 possess NTPase and RTPase activities (Bisaillon et al., 1997; Bisaillon & Lemay, 1997; Kim et al., 2004; Noble & Nibert, 1997a, b). The activity of both proteins appeared relevant for viral RNA transcription, replication, capping and other core functions. For avian reovirus, previous work indicates that protein A displays NTPase activity (Yin et al., 2002). Another protein, µA, which was considered to be a functional homologue of mammalian reovirus µ2 (Noad et al., 2006), based on the characteristic motifs for nucleotide binding (Noble & Nibert, 1997b), might possess similar enzymic activity. The µA protein, containing a more than 4.5 kDa peptide at the N terminus, was thus expressed in E. coli, and the purified protein was used for NTPase and RTPase activity assays. The expression levels obtained in soluble fraction were low, but sufficient for this investigation. During µA protein expression and purification, we also found that it was very difficult to obtain µA in soluble fraction, when the µA-encoding region was cloned within the NcoI/XhoI site of the pET28a vector (data not shown). The reason for this low solubility level was unclear, but might reflect that expression of µA as a fusion peptide which contained a 4.5 kDa peptide at its N terminus could increase the solubility of µA protein.
To rule out the possibility that the enzymic activity was attributable to a contaminant in the sample preparation, µA was treated at different temperatures prior to reaction with [-32P]ATP and the products of the reactions were analysed by TLC. The hydrolysis of [-32P]ATP by µA increased up to 40 °C and was almost inhibited at 55 °C. Since bacterial phosphatases are characteristically thermoresistant at 65 °C (Tomazic-Allen, 1991), our results suggest that the phosphatase activity observed was not due to contaminants. Furthermore, alanine substitutions were introduced in µA that we expected to reduce its NTPase activity, since the substitutions were located in a putative nucleotide-binding motif of µA (Noble & Nibert, 1997a). Mutant protein K408A/K412A µA, bearing these substitutions, showed the same features as µA during protein purification from soluble fractions and a similar pattern of peptide fragments generated by V8 protease partial digestion, but its NTPase activity was abolished. Thus, NTPase activity was attributable to µA.
Our estimated values for the kinetic properties of µA in NTP hydrolysis were similar to those of several other viral NTPases. For example, the KM for ATP hydrolysis by NTPase/RNA helicase NPH-II from vaccinia virus and reovirus µ2 have been reported as 1.2 mM (Gross & Shuman, 1996) and 2.5 mM (Kim et al., 2004), respectively; the kcat for ATP hydrolysis by NTPase/RTPase D1 from vaccinia virus and reovirus µ2 have been reported as 606 min–1 (Myette & Niles, 1996) or 830 min–1 (Kim et al., 2004), respectively.
NTPase activity has been identified for the gene products of several viruses such as vaccinia virus (Paoletti & Moss, 1974), yellow fever virus (Warrener et al., 1993), Japanese encephalitis virus (Kuo et al., 1996) and dsRNA viruses, including mammalian reovirus (Bisaillon et al., 1997; Bisaillon & Lemay, 1997; Noble & Nibert, 1997a, b), orbivirus (Ramadevi & Roy, 1998) and rotavirus (Vasquez-Del Carpio et al., 2006; Taraporewala et al., 1999). The NTPase activity of these proteins hydrolysed only the – phosphoanhydride bond of NTPs, because NDPs were the only products generated from [-32P]NTPs. Avian reovirus µA possesses similar enzymic activity, which hydrolyses all four NTPs. Thus, the nature of the activity for µA is very similar to that of mammalian reovirus protein µ2 (Noble & Nibert, 1997b) and other previously reported viral proteins as described above. Protein µA is thus a second component of avian reovirus that is associated with NTPase activity. In addition, we also demonstrated that µA possesses RTPase activity, and that this activity was abolished when µA was mutated. The results show that µA is capable of hydrolysing NTP and RNA substrates, and suggest that both activities might be carried out at the same active site, which is probably specific for triphosphorylated nucleotides, although no kinetic competition assays were made in this study. In this regard, it has been reported that these two activities are carried out at the same active site of several viruses, such as mammalian reovirus 1 (Bisaillon & Lemay, 1997) and µ2 (Kim et al., 2004), vaccinia virus capping enzyme (Myette & Niles, 1996; Yu & Shuman, 1996) and rotavirus NSP2 (Vasquez-Del Carpio et al., 2006). The functions of the enzymic activity of µA in the biology of avian reovirus are unknown, but the hydrolysis of NTPs by µA may be important for generating energy that is used during viral RNA synthesis.
Because of the facts that avian reovirus cores are structurally and functionally similar to mammalian reoviral cores, and that mammalian reoviral core proteins µ2 and 1 are associated with NTPase and RTPase activity (Bisaillon et al., 1997; Bisaillon & Lemay, 1997; Noble & Nibert, 1997a, b), in addition to A and µA (associated with NTPase activity), the NTP hydrolysis activity of other core proteins cannot be ruled out. The most likely candidate for such a protein would be avian reovirus protein A, encoded by the L1 genome segment of avian reovirus, which is the homologue of mammalian reovirus protein 1 (Noble & Nibert, 1997b).
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ACKNOWLEDGEMENTS |
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Received 25 September 2006;
accepted 21 February 2007.
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, Lysine positions that were substituted by alanine in this study.
