Activity and Regulation of Plant Antiviral Protein

植物已经进化出几种策略来逃避病毒的攻击和随后的传播。参与防御的一组蛋白质是核糖体失活蛋白（RIP）。RIP是从核糖体RNA的保守区域去除嘌呤碱基的酶。早期的研究表明，脱嘌呤抑制蛋白质翻译，由此产生的毒性被引用来解释RIP的抗病毒活性;也就是说，局部宿主细胞死亡将限制病毒传播。美洲商陆抗病毒蛋白（PAP）是美洲商陆（Phytolacca americana）合成的一种RIP，可抑制植物和动物病毒的复制。我们是第一个证明这种活性是由于病毒基因组RNA的脱嘌呤作用，这抑制了病毒生命周期的几个步骤。我们对PAP和其他RIPs的了解大部分来自它们在异源系统中的表达;对它们在天然植物中的功能知之甚少。例如，PAP在烟草中的表达产生病毒抗性植物;然而，它们表现出不期望的表型，包括生长受阻和不育。我们将此归因于对美洲商陆中PAP如何调节的了解不足。 我们研究计划的长期目标是了解植物在病毒感染后存活的机制。我们选择美洲商陆作为我们的模型，因为它具有广谱病毒抗性。 我们的短期目标是：1）确定PAP如何靶向RNA进行脱嘌呤作用; 2）了解美洲商陆中PAP基因表达如何调节。总之，这些目标将扩大我们的知识美洲商陆抗病毒机制和调节PAP。 为了解决第一个目标，我们将确定结合PAP的RNA的序列和结构。我们还将确定哪些细胞RNA被PAP脱嘌呤。将这些信息与鉴定与PAP相互作用的蛋白质配对，将揭示PAP是否作为蛋白质复合物的一部分发挥作用，这可能影响底物选择。 为了解决第二个目标，我们将通过测试其近端区域在表达报告基因测定中的活性来表征PAP的启动子。基于计算机模拟分析表明存在参与植物胁迫信号传导的顺式作用元件，我们认为PAP转录受防御相关蛋白的调节。我们将确定转录因子结合特定区域的近端启动子，以支持这一假设。我们还将研究PAP的5 'UTR内含子的功能，以测试它是否有助于PAP的转录控制。 总之，这些实验将有助于了解PAP如何作为一种抗病毒蛋白而不引起细胞毒性，以及它的表达是如何调节美洲商陆。虽然美洲商陆不是一种模式植物，但所收集的知识对PAP的农业应用以及理解RIPs在植物防御中的功能是有用的。

Plants have evolved several strategies to evade virus attack and subsequent spread. One group of proteins involved in defense is the ribosome inactivating proteins (RIPs). RIPs are enzymes that remove a purine base from a conserved region of ribosomal RNA. Early studies showed that depurination inhibited protein translation, and resulting toxicity was cited to explain the antiviral activity of RIPs; that is, localized host cell death would limit virus spread. Pokeweed antiviral protein (PAP) is a RIP synthesized by the pokeweed plant (Phytolacca americana) that inhibits the replication of plant and animal viruses. We were the first to demonstrate that this activity is due to depurination of viral genomic RNA, which inhibits several steps of the virus life cycle. Much of what we know about PAP and other RIPs comes from their expression in heterologous systems; little is known of their function in native plants. For example, expression of PAP in tobacco produces virus-resistant plants; however, they exhibit undesirable phenotypes including stunted growth and sterility. We attribute this to insufficient understanding of how PAP is regulated in pokeweed. The long-term goal of our research program is to understand the mechanisms that allow plants to survive virus infections. We chose pokeweed as our model because it has broad-spectrum virus resistance. Our short term objectives are: 1) to determine how PAP targets RNAs for depurination; 2) to understand how PAP gene expression is regulated in pokeweed. Together, these objectives will expand our knowledge of pokeweed antiviral mechanisms and regulation of PAP. To address the first aim, we will identify the sequences and structures of RNAs that bind PAP. We will also determine what cellular RNAs are depurinated by PAP. Pairing this information with identification of proteins that interact with PAP will reveal whether PAP functions as part of a protein complex, which could influence substrate selection. To address the second aim, we will characterize the promoter of PAP by testing the activity of its proximal region in expression reporter assays. Based on in silico analysis indicating the presence of cis-acting elements involved in signaling plant stress, we suggest that PAP transcription is regulated by defense related proteins. We will identify transcription factors that bind specific regions of the proximal promoter to support this hypothesis. We will also investigate the function of a 5' UTR intron of PAP to test whether it contributes to transcriptional control of PAP. Together these experiments will contribute to understanding how PAP functions as an antiviral protein without causing cytotoxicity, and how its expression is regulated in pokeweed. Though pokeweed is not a model plant, knowledge gleaned can be useful for the agricultural application of PAP, and in understanding the function of RIPs in plant defense.