REGULATION OF PROTEIN TOBACCO MOSAIC VIRUS RNA COMPLEXES

蛋白质烟草花叶病毒 RNA 复合物的调控

基本信息

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
项目类别：
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

项目摘要

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.

细胞之间的通讯和分子运输通常通过细胞间连接（称为间隙）进行。烟草花叶病毒 (TMV) 是研究最深入的植物病毒之一，其细胞间易位是由其运动蛋白 (MP) 介导的，建议形成复合物与基因组 TMV RNA 结合，并将它们靶向并通过植物细胞间连接，即胞间连丝。因此，这些复合物代表了一组病毒RNA分子，其目的是进行细胞间运输，并被排除在翻译和复制之外。 MP 与 TMV RNA 的关联可能可以解释这种功能抑制。最近，莫斯科合作者发现 MP-TMV RNA 在体外和缺乏胞间连丝的分离植物原生质体中受到翻译抑制。然而，这些复合物在植物组织中具有感染性，表明它们在通过胞间连丝通道后转化为可翻译形式。 MP-RNA 复合物具有翻译和复制能力的分子机制是什么？ TMV MP 在体内和体外均被磷酸化。 MP 磷酸化可能充当病毒细胞间运动、复制和翻译之间的调节开关。莫斯科小组最近的观察结果表明，MP 磷酸化使 MP-RNA 复合物可在体外翻译并感染分离的原生质体，这支持了这一观点。拟议的研究将通过寻求三个具体目标来研究 MP 磷酸化在 TMV 运动和感染调节中的作用： I. 表征体外形成的 MP-TMV RNA 复合物的结构并阐明其在 MP 磷酸化后的变化。将使用原子力显微镜和 RNA 足迹法分析 TMV RNA 与未磷酸化或磷酸化 MP 的复合物，以评估 MP 磷酸化如何影响受 MP 结合保护的 RNA 区域。 MP 磷酸化可通过蛋白激酶处理或在 MP 磷酸化位点使用带负电荷的取代突变来实现。二.分离病毒感染期间体内形成的 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 足迹分析进行检查。这些复合物中包含的特定磷酸化氨基酸残基将通过磷酸氨基酸分析和肽图谱来确定。三．研究磷酸化如何影响 MP-TMV RNA 复合物的生物活性。体外和体内形成的 MP-TMV RNA 复合物的生物活性将通过其在显微注射到烟叶叶肉后门控胞间连丝的能力来评估，并在体外、分离的原生质体和植物组织中进行复制和翻译。