A Geminivirus DNA Replication Protein - Programming Its Plant Host

9809953 Hanley-Bowdoin双生病毒是一类真核脱氧核糖核酸病毒，其基因组是单链的，在受感染的植物细胞的细胞核中通过滚环机制进行复制。它们的基因组很小，编码一种对它们的复制至关重要的蛋白质，并依赖于它们宿主的复制机制。这项研究的总体目标是确定双生病毒招募宿主DNA复制机制的机制。研究人员发现，双生病毒感染诱导宿主DNA合成蛋白--增殖细胞核抗原(PCNA)在终末分化的植物细胞中积聚。在感染组织中，增殖细胞核抗原RNA水平和启动子活性也升高，表明双生病毒可以激活静止植物细胞中编码DNA合成蛋白的基因的转录。重要的病毒复制蛋白AL1足以在转基因植物的分化细胞中诱导增殖细胞核抗原的积聚，因此可能类似于哺乳动物DNA病毒的肿瘤抗原蛋白。在动物系统中，DNA肿瘤病毒通过一种共同的机制在宿主中诱导复制机制。它们编码的蛋白质可以直接激活编码DNA合成蛋白的基因的转录，和/或与细胞周期调节因子相互作用，使静止的细胞能够进行DNA复制。研究人员将通过1)确定AL1与细胞周期调节因子视网膜母细胞瘤蛋白之间的相互作用是否是增殖细胞核抗原转录所必需的，2)询问AL1的多个结构域是否有助于转录激活增殖细胞核抗原，以及3)识别与AL1相互作用的其他植物蛋白来诱导增殖细胞核抗原的转录，从而确定AL1是否使用类似或不同的机制来诱导分化的植物细胞中的增殖细胞核抗原的转录。双生病毒是研究真核细胞DNA复制和细胞周期调控的优秀模式系统，这些领域在植物系统中还没有得到广泛的研究。该项目的最终目标是阐明双生病毒修饰和维持寄主植物处于病毒DNA复制的最佳状态的途径，并将这一途径与植物细胞周期和转录调控过程联系起来。这项研究将确定并开始描述双生病毒/宿主调控网络的组成部分。长期目标是了解在双生病毒感染和正常细胞过程中每个成分是如何发挥作用的。这些实验完全基于植物病毒和寄主成分，为直接研究植物细胞周期调控提供了难得的机会。它们还将增加我们对真核病毒改变细胞过程的基本机制的理解，并可能阐明植物和动物之间的基本相似和/或差异。

9809953 Hanley-Bowdoin Geminiviruses are a family of eukaryotic DNA viruses with single-stranded genomes that replicate through a rolling circle mechanism in nuclei of infected plant cells. They have small genomes, encode one protein that is essential for their replication, and rely on the replication machinery of their hosts. The overall goal of this research is to determine the mechanisms whereby geminiviruses recruit host DNA replication machinery. The investigator showed that geminivirus infection induces the accumulation of the host DNA synthesis protein, proliferating cell nuclear antigen (PCNA), in terminally differentiated plant cells. PCNA RNA levels and promoter activity are also elevated in infected tissue, demonstrating that geminiviruses can activate transcription of genes encoding DNA synthesis proteins in quiescent plant cells. The essential viral replication protein, AL1, is sufficient to induce the accumulation of PCNA in differentiated cells of transgenic plants, and thus may be analogous to the tumor antigen proteins of mammalian DNA viruses. In animal systems, DNA tumor viruses induce replication machinery in their host through a combination of common mechanisms. They encode proteins that directly activate transcription of genes encoding DNA synthesis proteins and/or interact with cell cycle regulatory factors to render quiescent cells competent for DNA replication. The investigator will determine whether AL1 uses similar or different mechanisms to induce PCNA transcription in differentiated plant cells by 1) determining whether interaction between AL1 and the cell cycle regulatory factor, retinoblastoma protein, is required for PCNA transcription, 2) asking whether multiple domains of AL1 contribute to PCNA transcriptional activation, and 3) identifying other plant proteins that interact with AL1 for PCNA induction. Geminiviruses are excellent model systems for studying eukaryotic DNA replication and cell cycle regulation, areas of study that have not been pursued extensively in plant systems. The ultimate g oals of the project are to elucidate the pathway whereby geminiviruses modify and maintain their host plants in an optimal state for viral DNA replication and to relate this pathway to plant cell cycle and transcriptional regulatory processes. The research will identify and begin to characterize components of the geminivirus/host regulatory network. The long term objectives are to understand how each component functions during geminivirus infection and in normal cellular processes. These experiments, which are based entirely on plant virus and host components, afford a rare opportunity to study plant cell cycle regulation directly. They will also increase our understanding of the basic mechanisms whereby eukaryotic viruses modify cellular processes and may illustrate fundamental similarities and/or differences between plants and animals.