Analysis of Nucleorhabdovirus Replication in Plant and Yeast Cells

摘要本研究旨在开发可用于植物横纹肌病毒感染基因解剖的系统。最近，对一些负链动物病毒家族进行分子遗传分析的条件得到了发展，这对我们对这些病毒的认识产生了巨大的影响。不幸的是，使用这些系统恢复病毒的效率非常低，而且它们的成功依赖于SYNV无法获得的几个关键要素，包括组织培养中的视觉斑块测定。因此，研究将探索两种新的替代策略，一种是在烟草愈伤组织中，一种是在酵母中，依靠严格的选择来恢复适合遗传分析的复制微小基因组。目的1将集中于构建自主复制的微型基因组，其中包含复制所需的基因和一个抗除草剂基因，用于选择支持微型基因组复制的植物愈伤组织细胞。这将使用与复制所需的聚合酶基因（N， P和L）的共转化，加上用于编码N， P和L基因的小基因组rna转录的磁带和一个可选择的标记。表达的病毒聚合酶蛋白应该与转录的小基因组rna相互作用，以“启动”小基因组衍生物的复制。这种策略应该提供愈伤组织培养的选择性生长，支持表达除草剂抗性基因的SYNV微小基因组复制子的复制，以便在除草剂bialaphos存在下进行选择。这些具有生物活性的小基因组随后将用于遗传分析，以提供有关SYNV复制活性的信息，并作为拯救更复杂的具有生物活性的SYNV衍生物的平台，该衍生物含有五种常见的横纹病毒基因和可选择的抗生素标记物，可用于区分被小基因组或病毒感染的细胞。还将进行实验，分离含有所有6个病毒基因的SYNV衍生物，以评估整个植物感染所需的因素。目的2将探索利用酵母的遗传分析能力来研究SYNV的复制，并分离复制所需的宿主基因。为此，将表达N， P和L聚合酶核心蛋白的现有酵母细胞系转化为能够表达可选择标记（URA3）的小基因组，以提供表达复制小基因组衍生物的酵母转化子的严格恢复。这一目标的实现将是创造具有生物活性的重组衍生物的关键一步，这些衍生物可用于分离复制SYNV所需的宿主成分。如果这些方法中的任何一种成功，我们对植物横纹肌病毒复制和发病机制的理解将大大加快，酵母选择方案的各个方面可能对其他负链病毒具有相当大的适用性。大量的横纹肌病毒引起严重的植物病害，但sonchus yellow net virus （SYNV）是唯一一种能够进行分子分析的植物横纹肌病毒。SYNV还具有几个独特的属性，使其对负链RNA动物病毒的感染过程进行比较分析具有吸引力。例如，SYNV的负链基因组和复制策略与引起人类、牲畜和野生动物重大疾病的动物横纹肌病毒和副粘病毒具有许多共同特征。然而，这些药物之间的显著差异是显而易见的，SYNV复制的核阶段是最有趣的特征之一。SYNV在这方面的复制也类似于一种远亲单甲负链病毒，即神经营养性博纳病病毒。因此，SYNV的感染过程与植物病毒和动物病毒都有相关性，因此，通过该病毒获得的知识将有助于更好地了解引起巨大农业和人类疾病的大量病毒的复制和病理所涉及的各种因素

MCB 9904810PI: -Andrew O. Jackson AbstractThis research is designed to develop systems that can be applied to genetic dissection of plant rhabdovirus infections. Conditions suitable for molecular genetic analyses have recently been developed for several negative-strand animal virus families, and these are having an enormous impact on our understanding of these viruses. Unfortunately, virus recovery using these systems is very inefficient, and their success relies on several key elements that are not available for SYNV, including visual plaque assays in tissue cultures. Therefore the research will investigate two novel alternative strategies, one in tobacco callus tissue and one in yeast, that rely on stringent selection for recovery of replicating minigenomes suitable for genetic analyses. Objective 1 will focus on construction of autonomously replicating minigenomes containing the genes required for replication plus a herbicide resistance gene to be used for selection of plant callus cells supporting the replication of the minigenome. This will use co-transformations with the polymerase genes (N, P, and L) that are required for replication, plus a cassette for transcription of minigenome RNAs encoding the N, P, and L genes and a selectable marker. The expressed viral polymerase proteins should interact with the transcribed minigenome RNAs to "jump start" replication of the minigenome derivative. This strategy should provide selective growth of callus cultures supporting replication of SYNV minigenome replicons that expresses a herbicide resistance gene for selection in the presence of the herbicide bialaphos. These biologically active minigenomes will subsequently be used for genetic analyses to provide information on the replicative activities of SYNV, and as platforms to rescue more complex biologically active SYNV derivatives containing the five common rhabdovirus genes and a selectable antibiotic marker that can be used for discrimination of cells infected with the minigenome or the virus. Experiments will also be carried out to isolate SYNV derivatives containing all six viral genes in order to evaluate the factors necessary for whole plant infections. Objective 2 will explore use of the genetic analysis capability of yeast to study replication of SYNV and to isolate host genes required for replication. For this purpose, an existing yeast cell line expressing the N, P, and L polymerase core proteins will be transformed with a minigenome capable of expressing a selectable marker (URA3) to provide stringent recovery of yeast transformants expressing replicating minigenome derivatives. Achievement of this objective will constitute a crucial step toward creating biologically active recombinant derivatives that can be used to isolate host components required for replication of SYNV. If either of these approaches is successful, our understanding of plant rhabdoviruses replication and pathogenesis will be accelerated enormously and aspects of the yeast selection scheme could have considerable applicability to other negative-strand viruses. A large number of rhabdoviruses cause serious plant diseases, but sonchus yellow net virus (SYNV) is the only well characterized plant rhabdovirus amenable to molecular analysis. SYNV also has several unique attributes that make it attractive for comparative analyses of the infection processes of negative-strand RNA animal virus counterparts. For example, the negative-strand genome and replication strategies of SYNV share many common features with those of animal rhabdoviruses and paramyxoviruses that cause significant human, livestock and wildlife diseases. However, striking differences are evident among these agents with the nuclear phase of SYNV replication being one of the most intriguing features. SYNV is also similar in this aspect of its replication to a distantly related monopartite minus-strand virus, the neurotrophic borna disease virus. Therefore, the infection processes of SYNV have relevance to both plant and animal viruses, so, the knowledge obtained with this virus will promote better understanding of the diverse factors involved in the replication and pathology of a large order of viruses that cause diseases with enormous agricultural and human