Studies on molecular interactions in two closely related plant viruses putatively using different mechanisms for cell-to-cell movement

雀麦花叶病毒(BMV)和豌豆褪绿斑驳病毒(CCMV)是关系密切的两种植物病毒，但鉴于其对外壳蛋白(CP)的需求，建议采用不同的细胞间运动机制。CCMV的CP缺陷变异株可在细胞间转移，而BMV的CP缺陷变异株则不能。为了确定病毒在细胞间运动中需要CP的病毒因子(S)，我们制备了几种嵌合病毒，如BMV(ΔCP-GFP)和CCMV(ΔCP GFP)，其中绿色荧光蛋白基因取代了CP。将3a运动蛋白(MP)基因在嵌合体之间交换，构建了BMV(C3a/ΔCP-GFP)和CCMV(B3a/ΔCP-GFP)。虽然BMV(ΔCP-gfp)没有在细胞间移动，而CCMV(ΔCP-gfp)发生了移动，但BMV(C3a/ΔCP-gfp)发生了移动，而CCMV(B3a/ΔCP-gfp)则没有移动，证明了新冠病毒3a基因决定了CP基因对病毒传播的要求。我们已经证明了BMV 3a在体外与单链(Ss)RNA结合。在这里，我证明了CCMV 3a也与ss-RNA协同结合，而不是序列特异性。为了检测BMV和CCMV的3a蛋白的小管形成能力，将这些蛋白作为与GFP的融合蛋白在原生质体中进行监测。在原生质体中，当接种编码3a-GFP融合的BMV或CCMV衍生物时，从其表面诱导出管状结构。总而言之，一个可能的解释是BMV和CCMV可能至少有两种运动机制。这两种病毒都可以通过管状结构，也可以以MP-RNA复合体的形式运输胞间连丝。GST下拉实验表明，BMV MP在体外能与其CP结合。要了解每种病毒的CP需求与每种MP与CP结合能力之间的关系，还需要进一步的研究。

Brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are closely related plant viruses but suggested to use different mechanisms for cell-to-cell movement in view of its coat protein (CP) requirement. CP-defective variants of CCMV move from cell to cell but those of BMV do not. To determine the viralfactor (s) involved in the CP requirement in viral cell-to-cell movement, we made several chimeric viruses, e.g. BMV (ΔCP-GFP) and CCMV (ΔCP GFP), where green fluorescent protein (GFP) gene was substituted for the CP. The 3a movement protein (MP) genes were exchanged between the chimeras to construct BMV (C3a/ΔCP-GFP) and CCMV (B3a/ΔCP-GFP). Although BMV (ΔCP-GFP) did not move from cell to cell and CCMV (ΔCP-GFP) moved, BMV (C3a/ΔCP-GFP) moved and CCMV (B3a/ΔCP-GFP) did not move, demonstrating that bromovirus 3a genes determine the requirement of the CP gene for viral spread. We have showed that BMV 3a binds single-stranded (ss) RNA in vitro. Here, I demonstrated that the CCMV 3a also binds ss-RNA cooperatively without sequence specificity. To examine tubule-forming capacity of the 3a proteins of BMV and CCMV, these proteins were monitored in protoplasts as fusion proteins with GFP. In protoplasts, tubular structures were induced from their surface, when they were inoculated with BMV or CCMV derivatives encoding 3a-GFP fusions. Together, one possible explanation is that BMV and CCMV could take at least two mechanisms for movement. Either virus may move through tubular structures as well as could traffic plasmodesmata as MP-RNA complex. GST pull-down assay showed that BMV MP binds its CP in vitro. Further studies are required to understand correlation between CP requirement of each virus and binding ability of each MP with CP.