Effects of Internal Cleavages and Mutations in the C-Terminal Region of NIa Protease of Turnip Mosaic Potyvirus on the Catalytic Activity

Abstract

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.

Characterization of a Novel Ser-cisSer-Lys Catalytic Triad in Comparison with the Classical Ser-His-Asp Triad

“…Several hydrolases of this family, including human ubiquitin C-terminal hydrolase (23), tobacco etch virus protease (24), and turnip mosaic potyvirus NIa (25), are known to contain a CysHis-Asp triad. Many papain-like cysteine protease family members utilize a Cys-His-Asn catalytic triad.…”

Potyviral NIa Proteinase, a Proteinase with Novel Deoxyribonuclease Activity

“…The reaction was terminated after 30 min by addition of 6 times loading dye and was monitored by electrophoresis on 0.7% agarose gel followed by ethidium bromide staining. Lanes 3,5,7,9, and 11 represent the reaction products of His-tagged NIa, NIa C151A, NIa H46A, NIa D81N, and NIa D81G proteinase, respectively; lanes 4,6,8,10, and 12 represent the reaction products of GST-fused NIa, NIa C151A, NIa H46A, NIa D81N, and NIa D81G proteinase, respectively; samples with heat-denatured His-tagged NIa and GST-NIa added as negative control are represented in lanes 1 and 2, respectively. B, phage DNA (50 g) was incubated with purified His-tagged NIa proteinase (5 g) in 20 mM Tris-HCl, pH 8.2, containing 1 mM MgCl 2 at 37°C for different times, and the reaction was stopped by addition of equal volumes of acid lanthanum reagent (20 mM La(NO 3 ) 3 in 0.2 N HCl). The precipitate was removed by centrifugation, and absorbance of the supernatant solution was measured at 260 nm against a blank sample without enzyme.…”

Proteinases Involved in Plant Virus Genome Expression

“…However, when the cleavage profiles of precursor and processed forms of the TEV NIa proteinase were analyzed, most substrates were processed in a similar fashion by all proteolytic forms. Further autoprocessing at specific positions of the NIa C-terminal region has been described in turnip mosaic potyvirus (TuMV) (Kim et al, 1995;MCnard et al, 1995;Kim et al, 1996) and TEV (Parks et al, 1995). Further autoprocessing at specific positions of the NIa C-terminal region has been described in turnip mosaic potyvirus (TuMV) (Kim et al, 1995;MCnard et al, 1995;Kim et al, 1996) and TEV (Parks et al, 1995).…”

“…Only at the 6K1-CI site, slight processing differences could be observed (Parks et al, 1992). Whereas a truncated TuMV 25K product (lacking the last 20 aa) was as active as the complete 27K proteinase for the cleavage at the 6K1-CI site (Kim et al, 1995), a TuMV 24K protein (lacking the last 30 aa) did not cleave at this site (Kim et al, 1996), and a TEV 25K protein (lacking the last 24 aa) was approximately onetwentieth as efficient in proteolysis of the NIb-CP site as the full-length form (Parks et al, 1995). The sequences around these cleavage sites were not similar to the typical heptapeptide recognition signals.…”