Regulation and antiviral mechanism of a plant ribosome inactivating protein

植物已经进化出几种策略来逃避病原体的攻击和随后的疾病。一组通常被认为与防御有关的蛋白质是核糖体失活蛋白。这些酶是由许多植物合成的从核糖体RNA的保守区域去除嘌呤碱基的酶。早期的研究表明，这种去嘌呤化如何减缓蛋白质翻译的速度，由此产生的毒性被用来解释其在防御中的作用；也就是说，局部宿主细胞死亡会限制病原体的传播。然而，对其在原生植物中的作用机制研究甚少。有趣的是，其中一种核糖体失活蛋白之所以被首次发现，是因为它具有抗病毒而非毒性。即，由美洲商陆植物（Phytolacca americana）合成的美洲商陆抗病毒蛋白（PAP）可以抑制几种动植物病毒的复制。我们是第一个证明这种活性是由于病毒基因组RNA的体内去嘌呤；因此，酶的底物并不局限于核糖体RNA。我们的研究计划的目标将建立在这一重要发现的基础上，以确定PAP在其原生美洲商陆和在其他异种植物中表达时的抗病毒分子机制。我们的短期研究目标有两个：1)确定PAP如何在不引起宿主细胞死亡的情况下靶向病毒rna。我们将在细胞中共定位PAP和病毒rna，并鉴定可能介导PAP活性的蛋白质。2)此外，我们想要确定PAP在美洲商陆中的表达是如何被调控的，以了解其在其原生系统中的作用。我们将通过表征PAP的启动子和我们最近发现的PAP特异性小RNA和假基因的功能来研究PAP表达的转录和转录后控制。总之，这些研究的结果将为提高作物对病毒的耐受性策略提供有用的信息，并将有助于我们研究计划的长期目标，即扩大植物自然防御策略的知识。

Plants have evolved several strategies to evade pathogen attack and subsequent disease. One group of proteins generally thought to be involved in defense is the ribosome inactivating proteins. These are enzymes synthesized by many plants that remove a purine base from a conserved region of ribosomal RNA. Early studies showed how this depurination slowed the rate of protein translation, and resulting toxicity was cited to explain a role in defense; that is, localized host cell death would limit pathogen spread. However, little attention has been paid to their mechanisms of action in their native plants. Interestingly, one of these ribosome inactivating proteins was first discovered because of its antiviral, rather than toxic, properties. Namely, pokeweed antiviral protein (PAP), synthesized by the pokeweed plant Phytolacca americana, inhibits the replication of several plant and animal viruses. We were the first to demonstrate that this activity is due to in vivo depurination of the viral genomic RNA; therefore, the enzyme's substrate is not limited to ribosomal RNA. The objectives of our research program will build on this important finding, to determine the molecular mechanisms by which PAP is antiviral in its native pokeweed and when expressed in other, heterologous plants. Our short-term research objectives are two-fold: 1) to determine how PAP targets viral RNAs without causing host cell death. We will co-localize PAP and viral RNAs in cells and identify proteins that may mediate PAP activity. 2) In addition, we want to determine how PAP expression is regulated in pokeweed, to understand its role in its native system. We will investigate the transcriptional and post-transcriptional control of PAP expression by characterizing its promoter and the functions of a PAP-specific small RNA and pseudogene that we have recently identified. Together, results from these studies will provide useful information to improve strategies for crop tolerance to viruses, and will contribute to the long term goal of our research program to expand knowledge of the natural defense strategies of plants.