1887

Abstract

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 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 Mg or Mn, but not Ca or Zn. 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.

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2007-06-01
2019-10-23
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