Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement.
Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement.
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DOI：
10.1371/journal.ppat.1004868
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发表时间：
2015-05
期刊：
影响因子：
6.7
通讯作者：
Ziegler-Graff V
中科院分区：
文献类型：
--
作者：
Smirnova E;Firth AE;Miller WA;Scheidecker D;Brault V;Reinbold C;Rakotondrafara AM;Chung BY;Ziegler-Graff V
黄病毒科的病毒具有约5.2至6.3 kb的正义RNA基因组，并且它们仅限于受感染植物的韧皮部。黄病毒属和Polerovirus属包括黄病毒科中除一种病毒外的所有病毒。它们共享一个共同的基因块，该基因块编码外壳蛋白（ORF 3）、运动蛋白（ORF 4）和外壳蛋白的羧基末端延伸（ORF 5）。据报道，这三种蛋白质都参与病毒在植物中的韧皮部特异性运动。所有这三种都是从一个亚基因组RNA sgRNA1翻译而来的。在这里，我们报告了一个新的短ORF的发现，称为ORF3a，sgRNA1的5'端附近编码。最初，通过对大量比对病毒序列中的序列变异进行统计分析来预测该ORF。ORF3a位于ORF3的上游，其翻译起始于非AUG密码子。用芜菁黄化病毒（TuYV）（一种polerovirus）进行ORF3a蛋白P3a的功能分析，其中ORF3a的翻译开始于ACG密码子。ORF3a在体外从对应于sgRNA1的转录物翻译，并且免疫检测测定证实了P3a在感染的原生质体和农杆菌接种的植物中的表达。阻止P3a表达或过表达P3a的突变不影响TuYV在原生质体或接种的拟南芥叶片中的复制，但阻止病毒在植物中的系统感染（长距离移动）。从单独的病毒或质粒载体表达P3a补充了缺乏ORF3a的TuYV突变体的运动。荧光蛋白融合的亚细胞定位研究表明，P3a是针对高尔基体和胞间连丝，支持病毒运动中的重要作用P3a。为了最大化编码能力，RNA病毒通常编码重叠基因并使用不寻常的翻译控制机制。植物病毒表达病毒在植物中移动所需的蛋白质，通常来自非规范翻译的开放阅读框（ORF）。经济上重要的黄病毒科中的病毒局限于韧皮部（维管）组织，可能是由于它们的专门的韧皮部特异性运动蛋白。这些蛋白质从一种病毒mRNA sgRNA1翻译而来，通过在一个以上的AUG密码子处起始以表达重叠基因，并通过终止密码子的核糖体通读。在这里，我们描述了另一个从sgRNA1翻译的基因，ORF3a。ORF3a的翻译起始于非标准（非AUG）起始密码子。我们发现ORF3a不是病毒基因组复制所必需的，而是病毒在植物中长距离移动所必需的。通过提供来自另一种病毒或质粒载体的ORF3a产物P3a，可以反式恢复运动功能。P3a定位于高尔基体和邻近胞间连丝，支持细胞间运动的作用。总之，我们使用了一个强大的生物信息学工具来发现一个隐藏的基因，其产物是韧皮部特异性植物病毒运动所必需的，揭示了多个水平的翻译控制，调节来自单个mRNA的四种蛋白质的表达。
Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5’ end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement. In order to maximize coding capacity, RNA viruses often encode overlapping genes and use unusual translational control mechanisms. Plant viruses express proteins required for movement of the virus through the plant, often from non-canonically translated open reading frames (ORFs). Viruses in the economically important Luteoviridae family are confined to the phloem (vascular) tissue, probably due to their specialized phloem-specific movement proteins. These proteins are translated from one viral mRNA, sgRNA1, via initiation at more than one AUG codon to express overlapping genes, and by ribosomal read-through of a stop codon. Here, we describe yet another gene translated from sgRNA1, ORF3a. Translation of ORF3a initiates at a non-standard (not AUG) start codon. We found that ORF3a is not required for viral genome replication, but is required for long-distance movement of the virus in the plant. The movement function could be restored in trans by providing the ORF3a product, P3a, from another viral or plasmid vector. P3a localizes in the Golgi apparatus and adjacent to the plasmodesmata, supporting a role in intercellular movement. In summary, we used a powerful bioinformatic tool to discover a cryptic gene whose product is required for movement of a phloem-specific plant virus, revealing multiple levels of translational control that regulate expression of four proteins from a single mRNA.
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影响因子：
5.4
作者：
Brown, CM;DineshKumar, SP;Miller, WA
通讯作者：
Miller, WA
影响因子：
2.7
作者：
CHALHOUB, BA;KELLY, L;LAPIERRE, HD
通讯作者：
LAPIERRE, HD
DOI：
10.1099/vir.0.042499-0
发表时间：
2012-07
期刊：
The Journal of general virology
影响因子：
--
作者：
Firth AE;Brierley I
通讯作者：
Brierley I
影响因子：
6.7
作者：
Amari K;Boutant E;Hofmann C;Schmitt-Keichinger C;Fernandez-Calvino L;Didier P;Lerich A;Mutterer J;Thomas CL;Heinlein M;Mély Y;Maule AJ;Ritzenthaler C
通讯作者：
Ritzenthaler C
影响因子：
3
作者：
Firth, AE;Brown, CM
通讯作者：
Brown, CM