Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis.
Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis.
复制标题
从克隆的 cDNA 中拯救植物负链 RNA 病毒：深入了解包膜植物病毒的运动和形态发生
DOI：
10.1371/journal.ppat.1005223
复制
发表时间：
2015-10
期刊：
影响因子：
6.7
通讯作者：
Li Z
中科院分区：
文献类型：
--
作者：
Wang Q;Ma X;Qian S;Zhou X;Sun K;Chen X;Zhou X;Jackson AO;Li Z
反向遗传学系统已经建立了所有主要群体的植物DNA和正链RNA病毒，和我们的理解，他们的感染周期和发病机制，极大地受益于使用这些方法。然而，迄今为止，技术上的困难阻碍了反向遗传学在植物负链RNA（NSR）病毒中的应用。在这里，我们报告的恢复感染性病毒的克隆cDNA的模式植物NSR，苦菜黄网弹状病毒（SYNV）。该过程涉及农杆菌介导的全长SYNV反基因组RNA的转录和核蛋白（N），磷蛋白（P），大聚合酶核心蛋白和RNA沉默的病毒抑制因子在本氏烟草植物中的共表达。核心蛋白表达的优化导致高达26%的重组SYNV（rSYNV）感染农杆菌渗入植物。通过在N和P基因之间插入绿色荧光蛋白（GFP）基因而工程化的报告病毒rSYNV-GFP能够在系统感染期间和在重复的植物到植物机械传代之后表达GFP。用rSYNV-GFP的缺失分析表明SYNV细胞到细胞的运动需要sc4蛋白，并表明未卷曲的核衣壳是感染性运动实体。缺失分析还表明，糖蛋白是不需要的全身感染，虽然糖蛋白突变体是有缺陷的病毒粒子形态发生。总之，我们已经开发了一个强大的反向遗传学系统SYNV，提供了关键的见解的形态发生和运动的包膜植物病毒。我们的研究也提供了一个模板，开发类似的系统，用于其他植物NSR病毒的反向遗传分析。反向遗传学是病毒生物学、病理学和生物技术应用基础研究的有力工具。虽然植物负链RNA（NSR）病毒由弹状病毒科、布尼亚病毒科、蛇夫病毒科和几个未归属属的成员组成，这些成员共同引起许多经济上重要的作物疾病，但不幸的是，一些技术困难阻碍了基因工程对这些病毒组的应用。本研究描述了第一个针对植物NSR病毒开发的反向遗传学系统。我们报道了一种从模式植物弹状病毒苦苣菜黄网病毒（SYNV）RNA的克隆cDNA生产感染性病毒的有效方法。我们还设计了一种重组SYNV载体，用于稳定表达荧光报告基因。使用这个系统，我们已经产生了有针对性的SYNV突变体，其分析提供了关键的见解包膜植物病毒的运动和形态发生过程。此外，我们的研究结果提供了一个模板，反向遗传学研究与其他植物弹状病毒，和策略，以规避技术困难，阻碍了这些应用到植物NSR病毒。
Reverse genetics systems have been established for all major groups of plant DNA and positive-strand RNA viruses, and our understanding of their infection cycles and pathogenesis has benefitted enormously from use of these approaches. However, technical difficulties have heretofore hampered applications of reverse genetics to plant negative-strand RNA (NSR) viruses. Here, we report recovery of infectious virus from cloned cDNAs of a model plant NSR, Sonchus yellow net rhabdovirus (SYNV). The procedure involves Agrobacterium-mediated transcription of full-length SYNV antigenomic RNA and co-expression of the nucleoprotein (N), phosphoprotein (P), large polymerase core proteins and viral suppressors of RNA silencing in Nicotiana benthamiana plants. Optimization of core protein expression resulted in up to 26% recombinant SYNV (rSYNV) infections of agroinfiltrated plants. A reporter virus, rSYNV-GFP, engineered by inserting a green fluorescence protein (GFP) gene between the N and P genes was able to express GFP during systemic infections and after repeated plant-to-plant mechanical passages. Deletion analyses with rSYNV-GFP demonstrated that SYNV cell-to-cell movement requires the sc4 protein and suggested that uncoiled nucleocapsids are infectious movement entities. Deletion analyses also showed that the glycoprotein is not required for systemic infection, although the glycoprotein mutant was defective in virion morphogenesis. Taken together, we have developed a robust reverse genetics system for SYNV that provides key insights into morphogenesis and movement of an enveloped plant virus. Our study also provides a template for developing analogous systems for reverse genetic analysis of other plant NSR viruses. Reverse genetics is a powerful tool for fundamental studies of virus biology, pathology and biotechnology applications. Although plant negative-strand RNA (NSR) viruses consist of members in the Rhabdoviridae, Bunyaviridae, Ophioviridae families and several unassigned genera that collectively account for many economically important crop diseases, unfortunately, several technical difficulties have hindered application of genetic engineering to these groups of viruses. This study describes the first reverse genetics system developed for plant NSR viruses. We report an efficient procedure for production of infectious virus from cloned cDNAs of sonchus yellow net virus (SYNV) RNAs, a model plant rhabdovirus. We have also engineered a recombinant SYNV vector for stable expression of a fluorescent reporter gene. Using this system, we have generated targeted SYNV mutants whose analyses provide key insights into enveloped plant virus movement and morphogenesis processes. Moreover, our findings provide a template for reverse genetics studies with other plant rhabdoviruses, and a strategy to circumvent technical difficulties that have hampered these applications to plant NSR viruses.