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Avian reovirus core protein µA expressed in Escherichia coli possesses both NTPase and RTPase activities

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

Analysis of the amino acid sequence of core protein µA of avian reovirus has indicated that it may share similar functions to protein µ2 of mammalian reovirus. Since µ2 displayed both nucleotide triphosphatase (NTPase) and RNA triphosphatase (RTPase) activities, the purified recombinant µA ( µA) was designed and used to test these activities. µA was thus expressed in bacteria with a 4.5 kDa fusion peptide and six His tags at its N terminus. Results indicated that µA possessed NTPase activity that enabled the protein to hydrolyse the – 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 k_cat 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.