Schaad M C, Haldeman-Cahill R, Cronin S, Carrington J C
Department of Biology, Texas A&M University, College Station, Texas 77843, USA.
J Virol. 1996 Oct;70(10):7039-48. doi: 10.1128/JVI.70.10.7039-7048.1996.
A mutational analysis was conducted to investigate the functions of the tobacco etch potyvirus VPg-proteinase (NIa) protein in vivo. The NIa N-terminal domain contains the VPg attachment site, whereas the C-terminal domain contains a picornavirus 3C-like proteinase. Cleavage at an internal site separating the two domains occurs in a subset of NIa molecules. The majority of NIa molecules in TEV-infected cells accumulate within the nucleus. By using a reporter fusion strategy, the NIa nuclear localization signal was mapped to a sequence within amino acid residues 40 to 49 in the VPg domain. Mutations resulting in debilitation of NIa nuclear translocation also debilitated genome amplification, suggesting that the NLS overlaps a region critical for RNA replication. The internal cleavage site was shown to be a poor substrate for NIa proteolysis because of a suboptimal sequence context around the scissile bond. Mutants that encoded NIa variants with accelerated internal proteolysis exhibited genome amplification defects, supporting the hypothesis that slow internal processing provides a regulatory function. Mutations affecting the VPg attachment site and proteinase active-site residues resulted in amplification-defective viruses. A transgenic complementation assay was used to test whether NIa supplied in trans could rescue amplification-defective viral genomes encoding altered NIa proteins. Neither cells expressing NIa alone nor cells expressing a series of NIa-containing polyproteins supported increased levels of amplification of the mutants. The lack of complementation of NIa-defective mutants is in contrast to previous results obtained with RNA polymerase (NIb)-defective mutants, which were relatively efficiently rescued in the transgenic complementation assay. It is suggested that, unlike NIb polymerase, NIa provides replicative functions that are cis preferential.
进行了突变分析以研究烟草蚀纹马铃薯Y病毒VPg蛋白酶（NIa）蛋白在体内的功能。NIa的N端结构域包含VPg附着位点，而C端结构域包含一个小RNA病毒3C样蛋白酶。在分离这两个结构域的内部位点的切割发生在一部分NIa分子中。烟草蚀纹病毒感染细胞中的大多数NIa分子在细胞核内积累。通过使用报告基因融合策略，将NIa核定位信号定位到VPg结构域中氨基酸残基40至49内的一个序列。导致NIa核转运减弱的突变也使基因组扩增减弱，这表明核定位信号与RNA复制关键区域重叠。由于可裂解键周围的序列环境不理想，内部切割位点被证明是NIa蛋白水解的不良底物。编码具有加速内部蛋白水解的NIa变体的突变体表现出基因组扩增缺陷，支持了缓慢的内部加工提供调节功能的假设。影响VPg附着位点和蛋白酶活性位点残基的突变导致扩增缺陷型病毒。使用转基因互补试验来测试反式提供的NIa是否可以拯救编码改变的NIa蛋白的扩增缺陷型病毒基因组。单独表达NIa的细胞或表达一系列含NIa多聚蛋白的细胞均不支持突变体扩增水平的增加。NIa缺陷型突变体缺乏互补作用，这与先前用RNA聚合酶（NIb）缺陷型突变体获得的结果相反，后者在转基因互补试验中得到了相对有效的拯救。有人提出，与NIb聚合酶不同，NIa提供顺式优先的复制功能。
