Kasschau K D, Carrington J C
Department of Biology, Texas A&M University, College Station 77843, USA.
Virology. 1995 May 10;209(1):268-73. doi: 10.1006/viro.1995.1254.
The helper component-proteinase (HC-Pro) of tobacco etch potyvirus (TEV) is a multifunctional protein with several known activities. The N-terminal region is required for aphid transmission and efficient genome amplification, the central region is required for long-distance movement in plants, and the C-terminal domain is a cysteine-type proteinase that autocatalytically cleaves between itself and the P3 protein. To investigate the requirement for HC-Pro-mediated proteolysis during viral replication, a variety of mutations resulting in amino acid substitutions and insertions in the proteolytic domain and cleavage site motif were introduced into the viral genome. Mutations affecting the active site residues, His722 and Cys649, or the cleavage site P1' residue, Gly764, inhibited proteolytic activity of HC-Pro. Mutant genomes containing these modifications were amplification-defective in protoplasts and plants. Mutants with substitutions affecting several conserved, but non-active site, residues (Ser610, Cys694, and Asp715) within the proteinase domain encoded active HC-Pro proteinases and were similar to parental virus in protoplasts and plants. To determine if the replication defect of the proteinase-debilitated mutants was due to inactivation of HC-Pro proteolytic activity or simply to the inability of HC-Pro and P3 protein to separate, a sequence coding for a heterologous cleavage site recognized by the TEV NIa proteinase was inserted between the HC-Pro and P3 coding regions of an active site mutant. This cleavage site was functional in vitro using purified NIa proteinase. However, this modification was insufficient to restore amplification activity to the mutant. In addition, the active site mutant was not complemented by wild-type HC-Pro supplied in trans by transgenic plants. These results suggest that an active HC-Pro proteinase is required in cis for TEV genome amplification.
烟草蚀纹马铃薯Y病毒（TEV）的辅助成分蛋白酶（HC-Pro）是一种具有多种已知活性的多功能蛋白。N端区域是蚜虫传播和高效基因组扩增所必需的，中央区域是植物长距离移动所必需的，而C端结构域是一种半胱氨酸型蛋白酶，可在其自身与P3蛋白之间进行自催化切割。为了研究病毒复制过程中HC-Pro介导的蛋白水解的必要性，将导致蛋白水解结构域和切割位点基序中氨基酸替换和插入的各种突变引入病毒基因组。影响活性位点残基His722和Cys649或切割位点P1'残基Gly764的突变抑制了HC-Pro的蛋白水解活性。含有这些修饰的突变基因组在原生质体和植物中扩增缺陷。蛋白酶结构域内影响几个保守但非活性位点残基（Ser610、Cys694和Asp715）的替换突变体编码有活性的HC-Pro蛋白酶，并且在原生质体和植物中与亲本病毒相似。为了确定蛋白酶功能减弱的突变体的复制缺陷是由于HC-Pro蛋白水解活性的失活还是仅仅由于HC-Pro和P3蛋白无法分离，在活性位点突变体的HC-Pro和P3编码区域之间插入了一个编码TEV NIa蛋白酶识别的异源切割位点的序列。该切割位点在体外使用纯化的NIa蛋白酶时具有功能。然而，这种修饰不足以恢复突变体的扩增活性。此外，活性位点突变体不能被转基因植物反式提供的野生型HC-Pro互补。这些结果表明，活性HC-Pro蛋白酶顺式存在是TEV基因组扩增所必需的。
