竹嵌紋病毒核酸複製酶中類解旋酵素區間motif III與motif V之功能分析

Abstract

Bamboo mosaic virus (BaMV) is a single-strand positive-sense RNA virus and its genome contains five open reading frames (ORFs). The 155-kDa viral replicase, encoded by ORF1, contains a central helicase-like domain according to the existence of several conserved motifs characteristic of RNA helicase superfamily 1 (SF1). Previous studies demonstrated that the helicase-like domain exhibits ATPase and 5’-triphosphatase activities. Also, the mutation Q826A at the motif V resulted in 17- and 80-fold decreases in value of Km and kcat, respectively, in the aspect of ATPase activity. The function of motif III and motif V of RNA SF1 is not well documented yet. To know more about it, conserved amino acids including D730 and Q733A at the motif III and T821, Q826, G827 and T829 at the motif V of BaMV were subjected to mutagenesis in this study. After mutations, the helicase-like domain was purified by Ni-NTA and gel filtration chromatography, which revealed the protein present in three oligomeric forms (monomer, dimer and polymers). Because the monomeric protein had the strongest ATPase activity, it was used in this study. As for ATPase activity, Q826S, Q826T and Q826H had 90- and 130-fold decreases in value of Km and kcat, respectively, similar to the effects brought by Q286A mutation. Mutations of T821A and T829A resulted in 30- and 100-fold decreases in value of Km and kcat, respectively. However, mutations of T821S and T829S only caused 3- and 8-fold decreases in value of Km and kcat. D730E, Q733A, Q733K, Q733R, G827A and G827V were unstable in the ATPase activity assay. In conclusion, the mutations at motif V of helicase-like domain of BaMV enhanced affinity to ATP but decreased catalytic efficiency.