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）和豇豆褪绿斑驳病毒（CCMV）是密切相关的植物病毒，但鉴于其对外壳蛋白（CP）的需求，建议使用不同的细胞间运动机制。CP缺陷的CCMV变异体在细胞间移动，而BMV则不然。为了确定在病毒细胞间移动中CP需要的病毒因子，我们制备了几种嵌合病毒，例如BMV（ΔCP-GFP）和CCMV（Δ CPGFP），其中绿色荧光蛋白（GFP）基因取代CP。在嵌合体之间交换3a运动蛋白（MP）基因以构建BMV（C3 a/ΔCP-GFP）和CCMV（B3 a/ΔCP-GFP）。尽管BMV（ΔCP-GFP）没有在细胞间移动，CCMV（ΔCP-GFP）移动，但BMV（C3 a/ΔCP-GFP）移动，而CCMV（B3 a/ΔCP-GFP）不移动，表明溴病毒3a基因决定了病毒传播对CP基因的需求。我们已经证明，BMV 3a结合单链（ss）RNA在体外。在这里，我证明了CCMV 3a也与ss-RNA协同结合，而没有序列特异性。为了检测BMV和CCMV的3a蛋白的微管形成能力，在原生质体中监测这些蛋白作为与GFP的融合蛋白。在原生质体中，当用编码3a-GFP融合体的BMV或CCMV衍生物接种时，从其表面诱导管状结构。总的来说，一种可能的解释是BMV和CCMV至少有两种运动机制。这两种病毒都可以通过管状结构移动，也可以作为MP-RNA复合物运输胞间连丝。GST pull-down实验表明BMV MP在体外与CP结合。需要进一步的研究来了解每种病毒的CP需求和每种MP与CP结合能力之间的相关性。

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.