RNA helicase activity of the plum pox potyvirus Cl protein expressed inEscherichia coli. Mapping of an RNA binding domain

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Abstract
The plum pox potyvirus (PPV) cylindrical inclusion (Cl) protein fused to the maltose binding protein (MBP) has been synthesized in Escherichia coli and purified, by affinity chromatography in amylose resin. In the absence ofany other viral factors, the fusion product had NTPase, RNA binding and RNA helicase activities. These in vitro activities were not affected by removal of the last 103 amino acids of the Cl protein. However, other deletions in the C-terminal part of the protein, although leaving intact all the region conserved in RNA helicases, drastically impaired the ability to unwind dsRNA and to hydrolyze NTPs. A mutant protein lacking the last 225 residues retained the competence to interact with RNA. Further deletions mapped boundaries of the RNA binding domain within residues 350 and 402 of the PPV Cl protein. This region includes the arginine-rich motif VI, the most carboxy terminal conserved domain of RNA helicases of the superfamily SF2. These results indicate that NTP hydrolysis is not an essential component for RNA binding of the PPV Cl protein.