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

Analysis of the amino acid sequence of core protein microampere of avian reovirus has indicated that it may share similar functions to protein mu2 of mammalian reovirus. Since mu2 displayed both nucleotide triphosphatase (NTPase) and RNA triphosphatase (RTPase) activities, the purified recombinant microampere (r microampere) was designed and used to test these activities. Microampere was thus expressed in bacteria with a 4.5 kDa fusion peptide and six His tags at its N terminus. Results indicated that r microampere possessed NTPase activity that enabled the protein to hydrolyse the beta-gamma 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 r microampere abolished NTPase activity, further suggesting that NTPase activity is attributable to protein r microampere. The activity of r microampere is dependent on the divalent cations Mg(2+) or Mn(2+), but not Ca(2+) or Zn(2+). Optimal NTPase activity of r microampere was achieved between pH 5.5 and 6.0. In addition, r microampere enzymic activity increased with temperature up to 40 degrees C and was almost totally inhibited at temperatures higher than 55 degrees C. Tests of phosphate release from RNA substrates with r microampere or K408A/K412A r microampere indicated that r microampere, but not K408A/K412A r microampere, displayed RTPase activity. The results suggested that both NTPase and RTPase activities of r microampere might be carried out at the same active site, and that protein microampere could play important roles during viral RNA synthesis.