Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology ICGEB, Aruna Asaf Ali Marg, New Delhi, 110067, India.
Arch Virol. 2013 Sep;158(9):1931-41. doi: 10.1007/s00705-013-1675-x. Epub 2013 Apr 11.
Geminiviruses replicate their single-stranded genomes with the help of only a few viral factors and various host cellular proteins primarily by rolling-circle replication (RCR) and/or recombination-dependent replication. AtRAD51 has been identified, using the phage display technique, as a host factor that potentially interacts with the Rep protein of mungbean yellow mosaic India virus (MYMIV), a member of the genus Begomovirus. In this study, we demonstrate the interaction between MYMIV Rep and a host factor, AtRAD51, using yeast two-hybrid and β-galactosidase assays, and this interaction was confirmed using a co-immunoprecipitation assay. The AtRAD51 protein complemented the rad51∆ mutation of Saccharomyces cerevisiae in an ex vivo yeast-based geminivirus DNA replication restoration assay. The semiquantitative RT-PCR and northern hybridization data revealed a higher level of expression of the Rad51 transcript in MYMIV-infected mungbean than in uninfected, healthy plants. Our findings provide evidence for a possible cross-talk between RAD51 and MYMIV Rep, which essentially controls viral DNA replication in plants, presumably in conjunction with other host factors. The present study demonstrates for the first time the involvement of a eukaryotic RAD51 protein in MYMIV replication, and this is expected to shed light on the machinery involved in begomovirus DNA replication.
双生病毒仅利用少数病毒因子和各种宿主细胞蛋白，主要通过滚环复制（RCR）和/或依赖重组的复制来复制其单链基因组。利用噬菌体展示技术，已鉴定出 AtRAD51 是一种宿主因子，它可能与豆黄斑驳印度病毒（MYMIV）的 Rep 蛋白相互作用，豆黄斑驳印度病毒是菜豆黄花叶病毒属（Begomovirus）的一个成员。在这项研究中，我们使用酵母双杂交和β-半乳糖苷酶测定证实了 MYMIV Rep 与宿主因子 AtRAD51 之间的相互作用，并且使用共免疫沉淀测定证实了这种相互作用。AtRAD51 蛋白在体外酵母双生病毒 DNA 复制恢复测定中补充了酿酒酵母 rad51∆突变体的功能。半定量 RT-PCR 和 northern 杂交数据显示，在感染 MYMIV 的绿豆中，Rad51 转录本的表达水平高于未感染的健康植物。我们的研究结果为 RAD51 和 MYMIV Rep 之间可能存在的交叉对话提供了证据，这种对话本质上控制了植物中的病毒 DNA 复制，可能与其他宿主因子一起。本研究首次证明了真核 RAD51 蛋白参与 MYMIV 复制，这有望阐明参与双生病毒 DNA 复制的机制。
