Analysis of the novel modalities of gene expression of the smallest (220 nucleotides) replicating virusoid

最小（220个核苷酸）复制类病毒基因表达的新模式分析

• 批准号: 6706-2008
• 负责人: AbouHaidar, Mounir
• 金额: $ 1.82万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2009
• 资助国家: 加拿大
• 起止时间: 2009-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=420299
• 关键词: Analysis; novel; modalities; gene; expression

Viroids and virusoids (viroid-like satellite RNAs) are small single-stranded (220-450 nucleotide) covalently closed circular (CCC) RNAs with no coding capacity for proteins (with the exception of the virusoid associated with the rice yellow mottle sobemovirus (scRYMV) which is the subject of this proposal) and are the smallest replicating circular RNA pathogens. They usually replicate through a "rolling circle" model producing larger concatemers which are then processed into monomeric forms either with a self-splicing hammerhead ribozyme (virusoids and viroids in the Avsunviroidae family) or by cellular enzymes. We reported (Collins et al, 1998.Virology 241:269-275) the characterization and sequence of this smallest scRYMV virusoid (220 nt) which depends on RYMV helper virus for replication and packaging. However, this is the smallest amongst all known viroids and virusoids and the only one to have an open reading (ORF) and codes for a basic 146/166 amino acid protein (16/18kD). Our goal is to unravel and comprehend the entire life cycle of this scRYMV. The objectives are: 1. Confirm the existence of 16/18 kD protein in RYMV infected rice by immunological assays and by mass spectroscopy. 2. Study the translation of this CCCRNA and in particular to demonstrate the internal initiation of translation of a Eukaryotic mRNA. 3. Determine the function (s) of this 16/18 kD protein and identify host proteins involved in the replication of a virusoid. 4. Study the initiation of replication of the CCCRNA and develop an in vitro functional system (RYMV RNA polymerase). Viral and host proteins involved in replication will be expressed in E. coli for replication of this virusoid. 5. Analyze the structure-function, pathogenesis and other novel properties of this virusoid. 6. Continue the investigation of degradation of E. coli RNAs by short double-stranded sequences from the scRYMV. Development of an E. coli model system to study the effects of double-stranded (DS) sequences on the degradation and regulation of genes (dicer-like effect and interference with micro RNAs (miRNAs)). Application of this E. coli system to elucidate the pathogenesis seen in infected plants with viruses, viroids and virusoids.

类病毒和类病毒（类病毒样卫星RNA）是小的单链（220-450核苷酸）共价闭合环状（CCC） RNA，没有蛋白质编码能力（与水稻黄斑病毒（scymv）相关的类病毒除外，这是本提案的主题），是最小的复制环状RNA病原体。它们通常通过“滚动循环”模式复制，产生较大的串联体，然后用自剪接锤头核酶（类病毒和类病毒属类病毒科）或细胞酶将其加工成单体形式。我们报道（Collins et al ., 1998）。这一最小的scymv类病毒（220 nt）依赖于RYMV辅助病毒进行复制和包装。然而，这是所有已知的类病毒和类病毒中最小的，也是唯一具有开放读取（ORF）并编码基本146/166氨基酸蛋白（16/18kD）的病毒。我们的目标是解开和理解这个scymv的整个生命周期。目标是：1。通过免疫分析和质谱分析，证实了RYMV感染水稻中存在16/ 18kd蛋白。2. 研究这个CCCRNA的翻译，特别是证明真核mRNA翻译的内部起始。3. 确定这个16/ 18kd蛋白的功能，并鉴定参与类病毒复制的宿主蛋白。4. 研究CCCRNA的起始复制，开发体外功能系统（RYMV RNA聚合酶）。参与复制的病毒和宿主蛋白将在大肠杆菌中表达以复制该类病毒。5. 分析该类病毒的结构功能、发病机制及其他新特性。6. 利用scymv的短双链序列继续研究大肠杆菌rna的降解。建立大肠杆菌模型系统，研究双链（DS）序列对基因降解和调控的影响（dicer-like效应和对微rna （miRNAs）的干扰）。应用该大肠杆菌系统阐明病毒、类病毒和类病毒感染植物的发病机制。