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

摘要

Analysis of the amino acid sequence of core protein mu A of avian reovirus has indicated that it may share similar functions to protein mu 2 of mammalian reovirus. Since mu 2 displayed both nucleotide triphosphatase (NTPase) and RNA triphosphatase (RTPase) activities, the purified recombinant mu A (r mu A) was designed and used to test these activities. mu A was thus expressed in bacteria with a 4.5 kDa fusion peptide and six His tags at its N terminus. Results indicated that r mu A 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 mu A abolished NTPase activity, further suggesting that NTPase activity is attributable to protein r mu A. The activity of r mu A is dependent on the divalent cations Mg2+ or Mn2+, but not Ca2+ or Zn2+. Optimal NTPase activity of r mu A was achieved between pH 5.5 and 6.0. In addition, r mu A 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 mu A or K408A/K412A r mu A indicated that r mu A, but not K408A/K412A r mu A, displayed RTPase activity. The results suggested that both NTPase and RTPase activities of r mu A might be carried out at the same active site, and that protein mu A could play important roles during viral RNA synthesis.