Studies on the mechanisms of cucumber mosaic virus RNA replication

黄瓜花叶病毒（CMV）和烟草花叶病毒（TMV）属于α-样正链RNA病毒组。在这项研究中，我们比较了两种病毒之间的病毒RNA复制的机制。首先，我们比较了使用膜浮选法的复制蛋白的亚细胞分级模式。对于CMV和TMV，复制蛋白被分成富含液泡膜的部分、富含内质网的部分和可溶性部分。第二，我们发现，除了纯化的液泡膜增强了复制TMV和CMV RNA在抽空烟草BY-2原生质体的无细胞提取物。这些结果表明液泡膜参与CMV和TMV RNA的复制。最后，我们探索了与CMV复制复合物相关的宿主蛋白。为此，我们构建了一个复制能力的CMV衍生物，携带FLAG标记的复制蛋白。我们从感染了这种CMV衍生物的BY-2原生质体中纯化膜，用去污剂溶解含有CMV复制复合物的膜，并用抗FLAG抗体缀合的琼脂糖珠纯化FLAG标记的复制蛋白。SDS-PAGE电泳分离纯化蛋白，银染鉴定。虽然结果仍然是初步的，几个特定的带中检测到FLAG纯化的馏分从原生质体感染的标记CMV衍生物。有趣的是，共纯化的宿主蛋白的模式是不同的，从那些与FLAG标记的TMV复制蛋白共纯化。这一结果表明CMV和TMV利用不同的宿主因子进行RNA复制。

Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV) belong to the alpha-like positive-strand RNA virus group. In this study, we compared the mechanisms of viral RNA replication between the two viruses. First, we compared the subcellular fractionation pattern of the replication proteins using the membrane-flotation method. Both for CMV and TMV, the replication proteins were fractionated into the tonoplast-rich fraction, endoplasmic reticulum-rich fraction and the soluble fraction. Second, we found that addition of purified vacuolar membranes enhanced the replication of TMV and CMV RNAs in cell-free extracts of evacuolated tobacco BY-2 protoplasts. These results suggest the involvement of vacuolar membranes in the replication of CMV and TMV RNAs. Lastly, we explored host proteins associated with the replication complex of CMV. To do this, we constructed a replication-competent CMV derivative that carried a FLAG-tagged replication protein. We purified membranes from BY-2 protoplasts infected with this CMV derivative, solubilized the membranes that contained the CMV replication complexes with a detergent, and purified the FLAG-tagged replication protein with anti-FLAG antibody-conjugated agarose beads. The co-purified proteins were separated with SDS-PAGE and detected by silver staining. Although the results are still preliminary, several specific bands were detected in the FLAG-purified fraction from protoplasts infected with the tagged CMV derivative. Interestingly, the pattern of co-purified host protein was different from those co-purified with FLAG-tagged TMV replication proteins. This result suggests CMV and TMV utilize different sets of host factors for RNA replication.