Effects of Mutations in the C-terminal Region of NIa Protease oncis-Cleavage between NIa and NIb

Abstract

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, I232D, or L235D mutation. The mutation of W212S, G213S, or I217D 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 glycines 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.

Overview and analysis of the polyprotein cleavage sites in the family Potyviridae

“…A second spontaneous internal cleavage site (Thr 207 /Ser 208 ) that removed the C‐terminal 36 amino acids abolished processing activity (Kim et al ., 1996). Mutation of the P4 Val to Asp or of the P1 Gln to Ala in the NIb/CP junction sequence (Thr 238 ‐Ala‐Val‐Tyr‐Ala‐Gln/Thr) greatly decreased processing but other changes in the region 218–237, including deletions (Lys 230 , Ser 229 ‐Lys 230 , Ser 229 ‐Leu 231 , or Ser 229 ‐Asp 234 ), insertions (5 × Gly at Ser 229 /Lys 230 or Ser 220 /Gln 221 ) and mutations (Phe 226 , Ile 232 or Leu 235 to Asp; Val 228 or Lys 230 to Glu), had little effect on the processing rate (Kim et al ., 1998). A genetic assay was used to examine the substrate specificity of the 6K1/CI junction (Pro‐Thr‐Val‐Tyr‐His‐Gln/Thr).…”