Correspondence

Analysis of the amino acid sequence of core protein mA of avian reovirus has indicated that it may share similar functions to protein m2 of mammalian reovirus. Since m2 displayed both nucleotide triphosphatase (NTPase) and RNA triphosphatase (RTPase) activities, the purified recombinant mA (rmA) was designed and used to test these activities. mA was thus expressed in bacteria with a 4.5 kDa fusion peptide and six His tags at its N terminus. Results indicated that rmA possessed NTPase activity that enabled the protein to hydrolyse the b–c 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 rmA abolished NTPase activity, further suggesting that NTPase activity is attributable to protein rmA. The activity of rmA is dependent on the divalent cations Mg2+ or Mn2+, but not Ca2+ or Zn2+. Optimal NTPase activity of rmA was achieved between pH 5.5 and 6.0. In addition, rmA enzymic activity increased with temperature up to 40 6C and was almost totally inhibited at temperatures higher than 55 6C. Tests of phosphate release from RNA substrates with rmA or K408A/K412A rmA indicated that rmA, but not K408A/K412A rmA, displayed RTPase activity. The results suggested that both NTPase and RTPase activities of rmA might be carried out at the same active site, and that protein mA could play important roles during viral RNA synthesis