Expression, Purification, and Identification of a Novel Self-Cleavage Site of the NIa C-Terminal 27-kDa Protease of Turnip Mosaic Potyvirus C5

Abstract

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.

Mutational analysis of the NIa protease from pepper vein banding potyvirus

“…In earlier studies, the activity of the recombinant NIa protease was assayed using a peptide containing the cleavage site and by the use of polyprotein substrates obtained by in vitro translation [5,17,18,22]. Hence, a nonapeptide (Ac-GGEVAHQAG-NH 2 ) corresponding to the cleavage site between the PVBV NIb and CP was synthesized, and was used as a substrate for the NIa protease.…”

“…The NIa proteases from Tobacco etch potyvirus (TEV) and Turnip mosaic potyvirus (TuMV) have been overexpressed and purified [17,22] and the recombinant proteases were shown to be catalytically active. However, a truncated NIa protease lacking the C-terminal 20-24 amino acids, resulting from autocatalytic activity of the recombinant protease, was less active [17,18,22].…”

“…However, a truncated NIa protease lacking the C-terminal 20-24 amino acids, resulting from autocatalytic activity of the recombinant protease, was less active [17,18,22]. Earlier mutational studies on TEV NIa protease indicated that His 46, Asp 81 and Cys 151 constitute the catalytic triad [11,14].…”

Metabolic engineering of plants using a disarmed potyvirus vector

“…Processing of the potyviral polyprotein seems to be a finely regulated process that produces the right amounts of the different gene products in time and space (Merits et al, 2002). Regulation is mainly based on the specific amino acid sequence recognized by the viral NIaPro, with some processing sites being cleaved faster than others Parks et al, 1995;Kim et al, 1995). Insertion of NIb in some of the intercistronic positions of the polyprotein may have fatal effects on this regulation.…”

“…Despite of their apparent simplicity of this genome expression strategy, the processing of potyviral polyprotein seems to be finely regulated resulting in the production of viral proteins in the right amount, time and space (Merits et al, 2002;Ivanov et al, 2014). The regulation is mainly based on the recognition efficiency of a viral encoded protease, NIaPro, responsible for the proteolytic processing of the main genome block of potyviruses from P3 to the end of the polyprotein Parks et al, 1995;Kim et al, 1995). Several studies demonstrated that there are some intercistronic positions that enable insertion of foreign genes without interrupting viral functions resulting in viable chimeric viruses.…”