REGULATION OF PROTEIN TOBACCO MOSAIC VIRUS RNA COMPLEXES

烟草花叶病毒（tobacco mosaic virus，TMV）是目前研究最多的植物病毒之一，它的细胞间转运是由其移动蛋白（movement protein，MP）介导的，MP与TMV RNA形成复合物，并通过植物细胞间的连接--胞间连丝（plasmodesmata）进行转运。 因此，这些复合物代表病毒RNA分子的库，这些病毒RNA分子注定用于细胞到细胞的运输，并且被排除在翻译和复制之外。 MP与TMV RNA的结合可能解释这种功能抑制。 最近，莫斯科的合作者发现，MP-TMV RNA在体外和缺乏胞间连丝的分离植物原生质体中被抑制。 然而，这些复合物在植物组织中变得具有感染性，这表明它们通过胞间连丝通道后转化为可翻译的形式。MP-RNA复合物能够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。 潜在地，MP磷酸化可以作为病毒细胞间移动与复制和翻译之间的调节开关。 莫斯科研究小组最近观察到MP的磷酸化使MP-RNA复合物在体外可翻译，并对分离的原生质体具有感染性，这支持了这一观点。 本研究拟从以下三个方面探讨MP磷酸化在TMV运动和侵染调控中的作用：I。研究MP与TMV RNA复合物的结构，并阐明MP磷酸化后复合物的结构变化。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磷酸化将通过蛋白激酶处理或使用MP磷酸化位点中的带负电荷的取代突变来实现。二.分离病毒感染时体内形成的MP-TMV RNA复合物，并对其结构和磷酸化程度进行表征。 将通过密度梯度离心从TMV感染的植物中分离MP-TMV RNA复合物，并通过Western印迹分析、原子力显微镜和RNA足迹法进行检查。 这些复合物中所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确定。三.研究磷酸化对MP-TMV RNA复合物生物活性的影响。 体外和体内形成的MP-TMV RNA复合物的生物活性将从它们在显微注射到烟草叶片叶肉中后门控胞间连丝的能力以及在体外、分离的原生质体和植物组织中进行复制和翻译的能力来评估。

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.