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

类病毒和类病毒(类病毒卫星RNA)是小的单链(220-450个核苷酸)共价闭合环(CCC)RNA，没有蛋白质编码能力(与水稻黄斑驳病毒(ScRYMV)相关的类病毒除外，该病毒是本提案的主题)，是最小的复制环状RNA病原体。它们通常通过“滚圈”模式进行复制，产生更大的串联体，然后通过自剪接锤头状核酶(Avsunviroidae家族中的类病毒和类病毒)或通过细胞酶加工成单体形式。我们报道了(Collins et al，1998.病毒学241：269-275)这个最小的scRYMV类病毒(220个核苷酸)的特征和序列，它依赖于RYMV辅助病毒的复制和包装。然而，这是所有已知的类病毒和类病毒中最小的，也是唯一一个具有开放读码(ORF)和编码基本146/166氨基酸蛋白质(16/18kD)的病毒。我们的目标是解开和理解这个SCRYMV的整个生命周期。本研究的目的是：1.用免疫学方法和质谱学方法证实RYMV侵染水稻中存在16/18kD蛋白。2.研究这种CCCRNA的翻译，特别是证明真核mRNA的翻译是由内部启动的。3.确定该16/18 kD蛋白的功能(S)，并鉴定参与类病毒复制的宿主蛋白。4.研究CCCRNA的复制起始，建立体外功能系统(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，以复制这种类病毒。5.对该病毒的结构功能、致病机理等新特性进行了分析。6.继续研究scRYMV的短双链序列对大肠杆菌RNA的降解作用。建立一个研究双链(DS)序列对基因降解和调控的影响(DICER样效应和干扰微小RNAs(MiRNAs))的大肠杆菌模型系统。应用这个大肠杆菌系统来阐明感染了病毒、类病毒和类病毒的植物的致病机理。

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.