White K A, Skuzeski J M, Li W, Wei N, Morris T J
School of Biological Sciences, University of Nebraska, Lincoln 68588-0118, USA.
Virology. 1995 Aug 20;211(2):525-34. doi: 10.1006/viro.1995.1434.
The plus-sense RNA genome of turnip crinkle virus (TCV) encodes at its 5' end a 28-kDa protein of unspecified function. Readthrough suppression of the p28 stop codon allows for the production of an 88-kDa product which is required for genome replication. Immunological analysis of the expression of p28 and p88 demonstrated that: (i) the genome directs the synthesis of polypeptides of approximately 28 and 88 kDa, (ii) the 88-kDa protein is immunologically related to p28, consistent with p88 being a readthrough product, and (iii) p28, but not p88, is detectable in vivo. An in vivo assay, in which readthrough is linked to the expression of a beta-glucuronidase reporter gene, showed that readthrough of the p28 amber stop codon occurs with an efficiency of approximately 1%. A similar efficiency of readthrough was observed when an altered context from the nonviable TCV mutant, mA2, containing a disrupted secondary structure (FfFa) spanning the p28 termination codon, was tested. This result suggests that the defective phenotype of mA2 is likely not linked to an alteration in readthrough efficiency. Additional studies demonstrated that complementation occurs in coinoculations with two nonviable TCV mutants, RT and APA, which are unable to express either p28 or p88, respectively. This result verifies that p28 is essential for TCV genome replication and provides the first definitive evidence for the role of a 5'-proximal open reading frame for any member of the family Tombusviridae.
芜菁皱缩病毒（TCV）的正义RNA基因组在其5'端编码一种功能未明的28 kDa蛋白。对p28终止密码子的通读抑制可产生一种88 kDa的产物，这是基因组复制所必需的。对p28和p88表达的免疫学分析表明：（i）基因组指导合成约28 kDa和88 kDa的多肽；（ii）88 kDa蛋白与p28存在免疫相关性，这与p88是通读产物一致；（iii）在体内可检测到p28，但检测不到p88。一项体内试验将通读与β-葡萄糖醛酸酶报告基因的表达联系起来，结果显示p28琥珀色终止密码子的通读效率约为1%。当测试来自无活力的TCV突变体mA2的改变后的序列时，观察到了类似的通读效率，mA2含有跨越p28终止密码子的破坏的二级结构（FfFa）。这一结果表明，mA2的缺陷表型可能与通读效率的改变无关。进一步的研究表明，在与两个无活力的TCV突变体RT和APA共接种时会发生互补作用，RT和APA分别无法表达p28或p88。这一结果证实了p28对TCV基因组复制至关重要，并为番茄丛矮病毒科任何成员的5'-近端开放阅读框的作用提供了首个确凿证据。
