Joseph J, Savithri H S
Department of Biochemistry, Indian Institute of Science, Bangalore.
Arch Virol. 2000;145(12):2493-502. doi: 10.1007/s007050070004.
The nuclear inclusion protein a (NIa) protease plays an important role in the life cycle of potyviruses by processing the viral polyprotein into functional proteins. For functional characterization, the NIa protease from Pepper vein banding potyvir s (PVBV) was overexpressed in Escherichia coli and purified. Using a recombinant polyprotein substrate containing the nuclear inclusion protein b (NIb)-coat protein (CP) cleavage site, a trans-cleavage assay was developed for the NIa protease. The polyprotein substrate also possessed the cleavage site between NIa and NIb, in addition to the NIb-CP site. However, no trans-cleavage by the NIa protease between NIa and NIb was detected indicating that the cleavage between NIa and NIb under natural conditions would be by a cis-cleavage reaction. Site-specific mutations of the conserved residues D81, D90, C110, T146, C151 and H167 were performed to investigate their roles in the catalytic process of the protease. Such an analysis has revealed that D81 and C151 constitute two of the catalytic triad residues in the NIa protease, D90 and C110 are not essential for catalysis, and T146 and H167 are probably involved in binding to Gln at the P1 position of the substrate.
核内含体蛋白a（NIa）蛋白酶通过将病毒多聚蛋白加工成功能蛋白，在马铃薯Y病毒属病毒的生命周期中发挥重要作用。为了进行功能表征，来自辣椒脉带病毒（PVBV）的NIa蛋白酶在大肠杆菌中过表达并纯化。利用含有核内含体蛋白b（NIb）-外壳蛋白（CP）切割位点的重组多聚蛋白底物，开发了一种针对NIa蛋白酶的反式切割分析方法。该多聚蛋白底物除了含有NIb-CP位点外，在NIa和NIb之间也具有切割位点。然而，未检测到NIa蛋白酶在NIa和NIb之间的反式切割，这表明在自然条件下NIa和NIb之间的切割是通过顺式切割反应进行的。对保守残基D81、D90、C110、T146、C151和H167进行了位点特异性突变，以研究它们在蛋白酶催化过程中的作用。这样的分析表明，D81和C151构成了NIa蛋白酶催化三联体中的两个残基，D90和C110对催化不是必需的，而T146和H167可能参与与底物P1位置的谷氨酰胺结合。
