Genome amplification and long-distance movement functions associated with the central domain of tobacco etch potyvirus helper component-proteinase
Genome amplification and long-distance movement functions associated with the central domain of tobacco etch potyvirus helper component-proteinase
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DOI：
10.1006/viro.1996.8368
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发表时间：
1997-02-17
期刊：
影响因子：
3.7
通讯作者：
Carrington, JC
中科院分区：
文献类型：
--
作者：
Kasschau, KD;Cronin, S;Carrington, JC
烟草蚀纹马铃薯Y病毒（TEV）辅助组分蛋白酶（HC-Pro）是一种多功能蛋白，参与蚜虫介导的传播、基因组扩增、多蛋白加工和长距离移动。为了研究这些功能中的三个之间的相互关系，将影响带电残基簇的25个丙氨酸扫描突变引入HC-Pro编码序列中。对突变体进行了体外HC-Pro蛋白水解活性、原生质体基因组扩增和烟草植株长距离运动等方面的分析。鉴定了三类突变体。I类突变体（共17个）能够进行基因组扩增、长距离移动和HC-Pro蛋白水解，其效率与亲本病毒相似。III类突变体（共1个）编码蛋白水解弱化的HC-Pro，并且是复制缺陷型的。II类突变体（共7个）编码蛋白水解活性的HC-Pro，但每个表现出抑制扩增表型，其特征在于在原生质体感染过程中的渐进关闭。II类突变体也表现出长距离运动的缺陷，积累到0至7.5%的相对水平在未接种的组织。野生型HC-Pro提供的反式能够部分拯救II类突变体的扩增缺陷的原生质体和长距离的运动缺陷的植物，虽然运动功能的互补的程度各不相同的突变体。七个II类突变中有六个影响了残基126和300之间的HC-Pro的中心区域，而只有一个影响了C-末端蛋白水解结构域。这些结果表明HC-Pro的中心区域对于有效的基因组扩增和长距离移动是必需的，并且参与这些过程的一个或多个HC-Pro功能至少部分是反式的。此外，长距离的运动性能的先前表征的HC-Pro缺陷型突变体（TEV-GUS/CCCE）的特点进一步使用嫁接非转基因和HC-Pro表达转基因植物。结果表明，HC-Pro在接种和未接种的组织中都是必需的，以补充TEV-GUS/CCCE运动缺陷。(C)北京：科学出版社.
The tobacco etch potyvirus (TEV) helper component-proteinase (HC-Pro, 460 amino acid residues) is a multifunctional protein involved in aphid-mediated transmission, genome amplification, polyprotein processing, and long-distance movement. To investigate the interrelationships between three of these functions, 25 alanine-scanning mutations affecting clusters of charged residues were introduced into the HC-Pro coding sequence. The resulting mutants were analyzed with respect to HC-Pro proteolytic activity in vitro, genome amplification in protoplasts, and long-distance movement in tobacco plants. Three classes of mutants were identified. Class I mutants (total of 17) were capable of genome amplification, long-distance movement, and HC-Pro proteolysis with efficiencies similar to parental virus. The class III mutant (total of 1) encoded a proteolytically debilitated HC-Pro and was replication-defective. Class II mutants (total of 7) encoded proteolytically active HC-Pro, but each exhibited a suppressed amplification phenotype that was characterized by a progressive shutoff during the course of infection in protoplasts. The class II mutants also exhibited defects in long-distance movement, accumulating to relative levels of 0 to 7.5% in noninoculated tissue. Wild-type HC-Pro supplied in trans was able to partially rescue the class II mutant amplification defects in protoplasts and long-distance movement defects in plants, although the extent of complementation of movement function varied for each mutant. Six of the seven class II mutations affected the central region of HC-Pro between residues 126 and 300, whereas only one affected the C-terminal proteolytic domain. These results indicate that the central region of HC-Pro is necessary for efficient genome amplification and long-distance movement, and that the one or more HC-Pro functions involved in these processes is at least partially trans-active. Additionally, the long-distance movement properties of a previously characterized HC-Pro-defective mutant (TEV-GUS/CCCE) were characterized further using grafted nontransgenic and HC-Pro-expressing transgenic plants. The results indicated that HC-Pro is required in both inoculated and noninoculated tissues to complement the TEV-GUS/CCCE movement defects. (C) 1997 Academic Press.