REGULATION OF PROTEIN TOBACCO MOSAIC VIRUS RNA COMPLEXES

蛋白质烟草花叶病毒 RNA 复合物的调控

• 批准号: 6351922
• 负责人: VITALY H CITOVSKY
• 金额: $ 2.52万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351922
• 关键词: Commonwealth of Independent States; atomic force microscopy; density gradient ultracentrifugation; genetic translation; intermolecular interaction; mutant; phosphoproteins; site directed mutagenesis; tobacco mosaic virus; virus RNA; virus protein; western blottings

Communication and molecular transport between cells often occur through intercellular connections, termed gap Cell-to-cell translocation of tobacco mosaic virus (TMV), one of the best studied plant viruses, is mediated by its movement protein (MP), proposed to form complexes with the genomic TMV RNA and target them to and through plant intercellular connections, the plasmodesmata. Thus, these complexes represent a pool of viral RNA molecules destined for cell-to-cell transport and excluded from translation and replication. Potentially, association of MP with TMV RNA may explain such functional inhibition. Recently, the Moscow collaborator has found that MP-TMV RNA are translationally repressed in vitro and in isolated plant protoplasts which lack plasmodesmata. However, these complexes became infectious in plant tissues, suggesting their conversion into a translatable form following passage through plasmodesmal channels. What is the molecular mechanism by which MP-RNA complexes become competent for translation and replication? TMV MP is phosphorylated both in vivo and in vitro. Potentially, MP phosphorylation may function as a regulatory switch between viral cell-to-cell movement and replication and translation. Recent observations by the Moscow group that phosphorylation of MP rendered MP-RNA complexes translatable in vitro and infectious to isolated protoplasts support this idea. The proposed research will study the role of MP phosphorylation in regulation of TMV movement and infection by seeking three specific objectives: I. To characterize the structure of in vitro-formed MP-TMV RNA complexes and elucidate its changes following MP phosphorylation. TMV RNA complexes with unphosphorylated or phosphorylated MP will be analyzed using atomic force microscopy and RNA footprinting to assess how MP phosphorylation affects RNA region(s) protected by MP binding. MP phosphorylation will be achieved either by protein kinase treatments or using negatively charged substitution mutations in the MP phosphorylation site. II. To isolate MP-TMV RNA complexes formed in vivo during viral infection and characterize their structure and degree of phosphorylation. MP-TMV RNA complexes will be isolated from TMV-infected plants by density gradient centrifugation and examined by Western blot analysis, atomic force microscopy, and RNA footprinting. The specific phosphorylated amino acid residues contained in these complexes will be determined by phosphoamino acid analysis and peptide mapping. III. To study how phosphorylation affects biological activity of MP-TMV RNA complexes. Biological activity of MP-TMV RNA complexes formed both in vitro and in vivo will be assessed from their ability to gate plasmodesmata following microinjection into tobacco leaf mesophyll and undergo replication and translation in vitro, in isolated protoplasts, and in plant tissues.

细胞之间的通信和分子转运通常是通过细胞间连接发生的，称为烟草镶嵌病毒（TMV）的间隙细胞到细胞转运，是研究植物病毒最佳的植物病毒之一，是由其运动蛋白（MP）介导的，该植物蛋白（MP）介导了与基因组TMV RNA形成基因组型rna并通过植物互构成植物互构成的配合物。 因此，这些络合物代表了一个注定用于细胞到细胞转运的病毒RNA分子池，并被排除在翻译和复制之外。 潜在地，MP与TMV RNA的关联可能解释了这种功能抑制作用。 最近，莫斯科合作者发现，MP-TMV RNA在体外被翻译抑制，并且在缺乏质量的植物原生质体中被抑制。 然而，这些复合物在植物组织中变得具有感染性，表明它们通过浆膜通道后转化为可翻译的形式。 MP-RNA复合物可以胜任翻译和复制的分子机制是什么？ TMV MP在体内和体外都被磷酸化。 可能，MP磷酸化可能是病毒细胞向细胞运动与复制和翻译之间的调节切换。 莫斯科小组最近观察到，MP的磷酸化使MP-RNA复合物在体外翻译，并具有传染性为分离的原生质体支持了这一想法。 拟议的研究将通过寻求三个特定目标来研究MP磷酸化在调节TMV运动和感染中的作用：I。表征体外形成的MP-TMV RNA复合物的结构，并在MP磷酸化后阐明其变化。将使用原子力显微镜和RNA足迹分析具有未磷酸化或磷酸化MP的TMV RNA复合物，以评估MP磷酸化如何影响受MP结合保护的RNA区域。 MP磷酸化将通过蛋白激酶处理或在MP磷酸化位点使用负电荷的取代突变来实现。 ii。分离在病毒感染过程中在体内形成的MP-TMV RNA复合物，并表征其结构和磷酸化程度。 MP-TMV RNA复合物将通过密度梯度离心从TMV感染的植物中分离出来，并通过蛋白质印迹分析，原子力显微镜和RNA足迹进行检查。 这些复合物中包含的特异性磷酸化氨基酸残基将通过磷酸氨基酸分析和肽映射确定。 iii。研究磷酸化如何影响MP-TMV RNA复合物的生物学活性。 在体外和体内形成的MP-TMV RNA复合物的生物学活性将从其在微分注射到烟草叶叶叶叶片后的栅极质体的能力，并在分离的原生质体和植物组织中进行体外复制和翻译。