The gene encoding the C-terminal protease domain (27 kDa) of the nuclear inclusion protein a of turnip mosaic potyvirus C5 was cloned and expressed as a fusion protein with glutathione S-transferase in Escherichia coli XL1-blue. Two forms of the protease (27 and 25 kDa) were purified from the fusion protein by glutathione affinity chromatography and Mono S chromatography and exhibited the specific proteolytic activity when a synthetic undecapeptide, Glu-Pro-Thr-Val-Tyr-His-Gln-Thr-Leu-Asn-Glu, or an in vitro translation product of the polyprotein containing the cleavage site between the nuclear inclusion protein b and the capsid protein, was used as a substrate. The purified proteases showed a Km of 1.15 +/- 0.16 mM and a Vmax of 0.74 +/- 0.091 mumol/mg/min with the synthetic peptide substrate. The 25-kDa protein was found to be generated by the cleavage between Ser223 and Gly224 near the C-terminus of the 27-kDa protease and to retain the specific proteolytic activity. The point mutation of Asp81 or Cys151, two putative active site residues in the 27-kDa protease, to Asn or Ser, respectively, prevented the generation of the 25-kDa protein and diminished the proteolytic activity of the protease drastically, suggesting that the 27-kDa protease cleaves itself between Ser223 and Gly224 to generate the 25-kDa protein.

The nuclear inclusion protein a (NIa) of turnip mosaic potyvirus is a protease processing the viral polyprotein into functional proteins. It has been shown that the NIa C-terminal 27-kDa protease cleaves itself between Ser-223 and Gly-224 to generate a 25-kDa protein lacking the C-terminal 20 amino acids. We have found a second internal cleavage near the C-terminus resulting in the degradation of the 25-kDa protein into a 24-kDa protein. Substitution of the active site Asp-81 or Cys-151 with Asn or Ser, respectively, prevented the second cleavage, suggesting that the internal cleavage is also due to the proteolytic activity of the NIa protease. This second internal cleavage was found to occur between Thr-207 and Ser-208, eliminating the C-terminal 36 amino acids from the 27-kDa protease. The proteolytic activity of the 24-kDa protein was not detected at all when it was measured using a nonapeptide containing the cleavage site between 6K1 and Cl as a substrate, suggesting that the C-terminal region between residues 208 and 223 contains essential amino acids for the processing of 6K1-Cl polyprotein. The deletion analyses of the C-terminal region revealed that at least 217 amino acids from the N-terminus are required for the catalytic activity of the NIa protease. The point mutation of Trp-212 to Ser, Gly-213 to Ser, or Ile-217 to Asp drastically abolished the catalytic activity, demonstrating that Trp-212, Gly-213, and Ile-217 are important for the processing of 6K1-Cl polyprotein.

Nuclear inclusion protein a (Nla) protease of turnip mosaic potyvirus is responsible for the processing of the viral polyprotein into functional proteins. The Nla protease was found to exhibit its optimum catalytic activity at approximately 15 degrees and a bell-shaped pH-dependent activity profile with a maximum at approximately pH 8.5. Kinetic studies showed that both Km and V(max) values were lower at 12 than at 25 degrees in all three different pH conditions, pH 7.0, 7.4, and 8.3, indicating that the higher activity at 12 degrees is due to the lower value of Km. Interestingly, the self-cleavage of the 27-kDa protease to generate the 25-kDa protease occurred more rapidly at 25 than at 12 degrees, implying that the C-terminal self-cleavage site may interfere with the binding of the peptide substrate to the active site of the protease. Mutations and deletions at the C-terminal cleavage site had no effect on the temperature dependence of the proteolytic activity, demonstrating that the C-terminal self-cleavage is not related to the low-temperature optimum catalytic activity. The fluorescence measurement of the Nla protease upon temperature variation revealed that the protease undergoes a large conformational change between 2 and 42 degrees and a drastic transition near 45 degrees, suggesting that the low-temperature optimum catalytic activity is due to the highly flexible structure of the Nla protease.

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.