REGULATION OF PROTEIN TOBACCO MOSAIC VIRUS RNA COMPLEXES

烟草花叶病毒(TMV)是研究最深入的植物病毒之一，其运动蛋白(MP)是由其运动蛋白(MP)介导的，它与TMV基因组RNA形成复合体，并通过胞间连接靶向TMV胞间连丝。因此，这些复合体代表了病毒RNA分子的池，目的是细胞间的运输，并排除在翻译和复制之外。MP与TMV RNA的结合可能解释了这种功能抑制。最近，莫斯科的这位合作者发现，MP-TMV RNA在体外和缺乏胞间连丝的分离植物原生质体中被翻译抑制。然而，这些复合体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的形式。MP-RNA复合体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的磷酸化可能作为病毒细胞间移动以及复制和翻译之间的调节开关发挥作用。莫斯科研究小组最近观察到，MP的磷酸化使MP-RNA复合体在体外可翻译，并对分离的原生质体具有感染性。本研究将通过三个具体目标来研究MP磷酸化在调控TMV运动和感染中的作用：1.表征体外形成的MP-TMV RNA复合体的结构，并阐明MP磷酸化后的变化。TMV与未磷酸化或磷酸化MP的RNA复合物将被原子力显微镜和RNA足迹分析，以评估MP磷酸化如何影响MP结合保护的RNA区域(S)。MP的磷酸化将通过蛋白激酶治疗或使用MP磷酸化位点上的负电荷替代突变来实现。分离病毒感染过程中在体内形成的MP-TMV RNA复合体，并对其结构和磷酸化程度进行表征。通过密度梯度离心法从感染TMV的植物中分离MP-TMV RNA复合体，并通过Western印迹分析、原子力显微镜和RNA足迹进行检测。这些复合体中含有的特定的磷酸化氨基酸残基将通过磷酸氨基酸分析和肽图来确定。研究磷酸化对MP-TMV RNA复合体生物活性的影响。在体外和体内形成的MP-TMV RNA复合体的生物活性将通过显微注射到烟草叶肉后对胞间连丝的门控能力以及在体外、在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.