Tobacco Etch Virus Protein P1 Traffics to the Nucleolus and Associates with the Host 60S Ribosomal Subunits during Infection
Tobacco Etch Virus Protein P1 Traffics to the Nucleolus and Associates with the Host 60S Ribosomal Subunits during Infection
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DOI：
10.1128/jvi.00928-14
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发表时间：
2014-09-01
影响因子：
5.4
通讯作者：
Daros, Jose-Antonio
中科院分区：
文献类型：
--
作者：
Martinez, Fernando;Daros, Jose-Antonio
马铃薯Y病毒属包括一大组正链RNA植物病毒，其基因组编码由三种病毒蛋白酶加工的大的多蛋白。P1蛋白是多聚蛋白的最氨基末端产物，是刺激病毒基因组扩增的辅助因子，其在感染过程中的作用尚不清楚。我们用烟草蚀纹病毒（TEV;马铃薯Y病毒属）克隆感染植物，其中P1用荧光蛋白标记以跟踪其表达和亚细胞定位，或用亲和标记以鉴定参与P1在感染过程中也参与的复合物的宿主蛋白。我们的研究结果表明，TEV P1只在感染的早期阶段在感染的细胞中积累，并且该蛋白质在感染期间显示动态的亚细胞定位、进出细胞核和核仁的运输。在核仁内，P1特别针对致密颗粒成分。因此，我们在TEV P1序列中发现了功能性的核仁定位和核输出信号。我们的研究结果还表明，TEV P1与宿主80 S胞质核糖体的物理相互作用，并在感染过程中特异性地结合到60 S核糖体亚基。报告蛋白的体外翻译测定表明，TEV P1刺激蛋白质翻译，特别是当从TEV内部核糖体进入位点驱动时。这些体外试验还表明，TEV辅助组分蛋白酶（HC-Pro）抑制蛋白质翻译。基于这些发现，我们提出，TEV P1刺激病毒蛋白在感染细胞中的翻译。重要在这项工作中，我们研究了烟草蚀纹病毒P1蛋白酶在感染过程中的作用。P1是马铃薯Y病毒属最神秘的蛋白质，马铃薯Y病毒属是一组感染植物的RNA病毒。我们的实验表明，病毒P1蛋白在感染的早期阶段仅在感染细胞中积累，并进出感染细胞的细胞核，特别是靶向核仁。我们的实验还表明，P1蛋白在感染过程中与宿主核糖体结合。基于这些发现和其他体外实验，我们提出P1蛋白在感染过程中刺激病毒蛋白的翻译。
The genus Potyvirus comprises a large group of positive-strand RNA plant viruses whose genome encodes a large polyprotein processed by three viral proteinases. P1 protein, the most amino-terminal product of the polyprotein, is an accessory factor stimulating viral genome amplification whose role during infection is not well understood. We infected plants with Tobacco etch virus (TEV; genus Potyvirus) clones in which P1 was tagged with a fluorescent protein to track its expression and subcellular localization or with an affinity tag to identify host proteins involved in complexes in which P1 also takes part during infection. Our results showed that TEV P1 exclusively accumulates in infected cells at an early stage of infection and that the protein displays a dynamic subcellular localization, trafficking in and out of the nucleus and nucleolus during infection. Inside the nucleolus, P1 particularly targets the dense granular component. Consistently, we found functional nucleolar localization and nuclear export signals in TEV P1 sequence. Our results also indicated that TEV P1 physically interacts with the host 80S cytoplasmic ribosomes and specifically binds to the 60S ribosomal subunits during infection. In vitro translation assays of reporter proteins suggested that TEV P1 stimulates protein translation, particularly when driven from the TEV internal ribosome entry site. These in vitro assays also suggested that TEV helper-component proteinase (HC-Pro) inhibits protein translation. Based on these findings, we propose that TEV P1 stimulates translation of viral proteins in infected cells.IMPORTANCE In this work, we researched the role during infection of tobacco etch virus P1 protease. P1 is the most mysterious protein of potyviruses, a relevant group of RNA viruses infecting plants. Our experiments showed that the viral P1 protein exclusively accumulates in infected cells at an early stage of infection and moves in and out of the nucleus of infected cells, particularly targeting the nucleolus. Our experiments also showed that P1 protein binds host ribosomes during infection. Based on these findings and other in vitro experiments we propose that P1 protein stimulates translation of viral proteins during infection.