REGULATION OF PROTEIN TOBACCO MOSAIC VIRUS RNA COMPLEXES

细胞间的通讯和分子运输通常通过细胞间连接进行，烟草花叶病毒（TMV）被称为间隙细胞-细胞易位，是研究得最好的植物病毒之一，它的运动蛋白（MP）介导其与基因组TMV RNA形成复合物，并将其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群病毒RNA分子，它们被排除在翻译和复制之外，用于细胞间的转运。潜在地，MP与TMV RNA的关联可能解释了这种功能抑制。最近，莫斯科合作者发现MP-TMV RNA在体外和分离的缺乏间连丝的植物原生质体中被翻译抑制。然而，这些复合物在植物组织中具有传染性，表明它们通过胞浆原丝通道转化为可翻译的形式。MP-RNA复合物具有翻译和复制能力的分子机制是什么？TMV MP在体内和体外都被磷酸化。潜在地，MP磷酸化可能在病毒细胞间运动、复制和翻译之间起调节开关的作用。莫斯科小组最近的观察结果表明，MP的磷酸化使MP- rna复合物在体外可翻译，并能感染分离的原生质体，这支持了这一观点。本研究将研究MP磷酸化在TMV运动和感染调控中的作用，寻求三个具体目标：1 .表征体外形成的MP-TMV RNA复合物的结构，并阐明MP磷酸化后其变化。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磷酸化可以通过蛋白激酶处理或在MP磷酸化位点使用带负电荷的替代突变来实现。2。分离MP-TMV病毒感染过程中在体内形成的RNA复合物，并对其结构和磷酸化程度进行表征。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Western blot分析、原子力显微镜和RNA足迹检测。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。3。研究磷酸化对MP-TMV RNA复合物生物活性的影响。MP-TMV RNA复合物在体外和体内形成的生物活性将通过其在烟草叶片叶肉中微注射后连接胞间连丝的能力以及在体外、分离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.