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)属于类α-正链RNA病毒群。在这项研究中，我们比较了两种病毒之间的病毒RNA复制机制。首先，我们用膜浮选法比较了复制蛋白的亚细胞分级模式。CMV和TMV的复制蛋白均分为富含液泡膜的部分、富含内质网的部分和可溶性部分。其次，我们发现，加入纯化的液泡膜可以促进烟草BY-2原生质体无细胞提取液中TMV和CMV RNA的复制。这些结果表明，液泡膜参与了CMV和TMV RNA的复制。最后，我们探索了与CMV复制复合体相关的宿主蛋白。为此，我们构建了一种携带标记复制蛋白的具有复制能力的CMV衍生物。我们从感染该CMV衍生物的BY-2原生质体中提纯了膜，用洗涤剂溶解了含有CMV复制复合体的膜，并用抗FLAG抗体偶联的琼脂糖珠纯化了FLAG标记的复制蛋白。共纯化的蛋白用SDS-PAGE分离，银染检测。虽然结果仍然是初步的，但在标记的CMV衍生物感染的原生质体的FLAG纯化组分中检测到了几条特异的条带。有趣的是，共纯化的宿主蛋白与标记TMV复制蛋白的共纯化的宿主蛋白的模式不同。这一结果表明CMV和TMV利用不同的寄主因子进行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.