Activity and Regulation of Plant Antiviral Protein

植物已经进化出几种策略来躲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(RIPs)。RIPs是从核糖体RNA的保守区域移除嘌呤碱基的酶。早期的研究表明，脱嘌呤抑制了蛋白质的翻译，由此产生的毒性被用来解释RIPS的抗病毒活性；也就是说，局部的宿主细胞死亡将限制病毒的传播。商陆抗病毒蛋白(PAP)是商陆植物(Phytolacca Americana)合成的抑制动植物病毒复制的RIP。我们是第一个证明这种活性是由于病毒基因组RNA的脱氨所致，这抑制了病毒生命周期的几个步骤。我们对PAP和其他RIPs的了解大多来自它们在异源系统中的表达；对它们在本地植物中的功能知之甚少。例如，在烟草中表达PAP产生了抗病毒的植株；然而，它们表现出不受欢迎的表型，包括生长受阻和不育。我们将此归因于对PAP如何在商陆中受到调控的了解不足。我们研究计划的长期目标是了解使植物在病毒感染下存活的机制。我们选择商陆作为我们的模型，是因为它具有广谱的病毒抗性。我们的短期目标是：1)确定PAP如何靶向RNA进行去嘌呤；2)了解PAP基因在商陆中的表达是如何调控的。总之，这些目标将扩大我们对商陆抗病毒机制和PAP调控的了解。为了解决第一个目标，我们将鉴定与PAP结合的RNA的序列和结构。我们还将确定哪些细胞RNA被PAP净化。将这些信息与与PAP相互作用的蛋白质的鉴定配对将揭示PAP是否作为蛋白质复合体的一部分发挥功能，这可能会影响底物选择。为了解决第二个目标，我们将通过在表达报告分析中测试PAP近端区域的活性来表征PAP的启动子。基于电子分析表明存在参与植物逆境信号转导的顺式作用元件，我们认为PAP转录受防御相关蛋白的调控。我们将确定结合近端启动子特定区域的转录因子来支持这一假说。我们还将研究PAP的5‘非编码区内含子的功能，以测试它是否有助于PAP的转录调控。这些实验将有助于理解PAP如何在不引起细胞毒性的情况下作为一种抗病毒蛋白发挥作用，以及它在商陆中的表达是如何调节的。虽然商陆不是模式植物，但所获得的知识有助于PAP的农业应用，并有助于理解RIPs在植物防御中的功能。

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.