A Geminivirus DNA Replication Protein - Programming Its Plant Host

Hanley-Bowdoin双病毒是一类真核DNA病毒，具有单链基因组，在受感染植物细胞的细胞核中通过滚圈机制进行复制。它们的基因组很小，编码一种对自身复制至关重要的蛋白质，并依赖宿主的复制机制。本研究的总体目标是确定双病毒招募宿主DNA复制机制的机制。研究人员发现，双病毒感染诱导宿主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累。感染组织中的PCNA RNA水平和启动子活性也升高，表明双病毒可以激活静止植物细胞中编码DNA合成蛋白的基因转录。在转基因植物的分化细胞中，必需的病毒复制蛋白AL1足以诱导PCNA的积累，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肿瘤病毒通过一系列共同机制在宿主体内诱导复制机制。它们编码的蛋白质直接激活编码DNA合成蛋白的基因转录和/或与细胞周期调节因子相互作用，使静止细胞能够进行DNA复制。研究者将确定AL1是否使用相似或不同的机制诱导分化植物细胞中的PCNA转录：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需要PCNA转录，2)询问AL1的多个结构域是否有助于PCNA转录激活，以及3)鉴定与AL1相互作用诱导PCNA的其他植物蛋白。双病毒是研究真核DNA复制和细胞周期调控的优秀模型系统，这些研究领域尚未在植物系统中广泛开展。该项目的最终目标是阐明双病毒修饰和维持其宿主植物在病毒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节过程联系起来。该研究将确定并开始表征双病毒/宿主调控网络的组成部分。长期目标是了解每种成分在双病毒感染期间和正常细胞过程中的作用。这些实验完全基于植物病毒和宿主成分，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修饰细胞过程的基本机制的理解，并可能说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.