Journal of Virology, December 2001, p. 12114-12120, Vol. 75, No. 24
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.24.12114-12120.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Graduate Institute of Agricultural Biotechnology, National Chung Hsing University, Taichung, Taiwan 40227, Republic of China
Received 8 June 2001/Accepted 18 September 2001
Open reading frame 1 (ORF1) of potexviruses encodes a viral
replicase comprising three functional domains: a capping enzyme at the
N terminus, a putative helicase in the middle, and a polymerase at the
C terminus. To verify the enzymatic activities associated with the
putative helicase domain, the corresponding cDNA fragment from bamboo
mosaic virus (BaMV) was cloned into vector pET32 and the protein was
expressed in Escherichia coli and purified by metal
affinity chromatography. An activity assay confirmed that the putative
helicase domain has nucleoside triphosphatase activity. We found that
it also possesses an RNA 5'-triphosphatase activity that specifically
removes the
phosphate from the 5' end of RNA. Both enzymatic
activities were abolished by the mutation of the nucleoside
triphosphate-binding motif (GKS), suggesting that they have a common
catalytic site. A typical m7GpppG cap structure was formed
at the 5' end of the RNA substrate when the substrate was treated
sequentially with the putative helicase domain and the N-terminal
capping enzyme, indicating that the putative helicase domain is truly
involved in the process of cap formation by exhibiting its RNA
5'-triphosphatase activity.
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