Mutational analyses were carried out to investigate whether the nuclear inclusion protein a (NIa) C-terminal amino acids of turnip mosaic potyvirus play any roles in the cis-cleavage between NIa and NIb. The processing rate of the NIa-NIb junction sequence was decreased significantly by either V240D or Q243A mutation while little affected by F226D, V228E, K230E, 1232D, or L235D mutation. The mutation of W212S, G213S, or 1217D abolishing the cleavage at the NIb-CP or 6K1-cylindrical inclusion protein junction sequence decreased the processing rate to half the level of that of the wild type. Deletion of the C-terminal one (K230), two (S229 and K230), three (S229 to L231), or six amino acids (S229 to D234) as well as the insertion of five giycines between S229 and K230 or between S220 and Q221 did not affect significantly the cleavage while the deletion of 20 amino acids (Q218 to S237) decreased the processing rate to 73% of that of the wild type. These results rule out the possibility that the C-terminal region plays a role as a spacer in right placement of the NIa-NIb junction sequence and demonstrate that the C-terminal 20 amino acids from Q218 to S237 are not crucial for the cis-cleavage of the NIa-NIb junction sequence.
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We thank Sung Hoon Back and Bum Suk Ham at POSTECH for the technical assistance of the in vitro transcription and in vitro translation of the proteins. This work was supported partly by the Center for Biofunctional Molecules, the HAN Project from Ministry of Science and Technology, and the R & D Promotion Center for Agriculture, Forestry and Fishery in Korea.