REGULATION OF CAP-INDEPENDENT TRANSLATION IN PLUS-STRAND RNA VIRUSES

该项目将通过让大学本科生、博士研究生和博士后研究人员参与研究来解决病毒如何进行其最重要的活动之一——合成所需蛋白质，从而造福社会。这项研究将在科学会议上发表，用于生物学专业的生物学入门教学，并用于向马里兰大学和鲍伊州立大学的本科生介绍旨在研究病毒如何以以前认为不会发生的方式操纵其基因组的尖端程序。此外，这项研究为一个过程（蛋白质合成）增加了一个新的理解维度，这个过程被认为主要是通过探索病毒生物学的一个关键方面的可能调控来解决的：它们如何从制造蛋白质的模板转变为通过改变模板与特定蛋白质的相互作用来复制模板。通过研究三种不同的病毒，它们似乎都使用相同的过程来控制蛋白质的产生，结果应该对一般的病毒研究产生广泛的影响。因此，这项研究的结果不仅适用于植物病毒，也适用于动物和人类病毒，包括那些导致动物和植物严重疾病的病毒。最近的研究结果表明，位于植物病毒3' utr中的翻译增强子需要清除结合的翻译因子/核糖体才能进行模板复制。PEMV包含三个3'cap-independent translation enhancer (3'CITEs): kl-TSS， PTE和TSS，其中只有两个被基因组RNA使用（kl-TSS/PTE），而三个都被亚基因组RNA使用。在Aim 1中，SHAPE RNA结构探测和诱变将用于确定gRNA独有的序列是否会改变TSS的结构以阻止其功能。在Aim 2中，将使用补偿性突变和SHAPE来检查新发现的元件（CAS）在三种病毒中阻断翻译因子与下游3' CITEs结合的能力。新发现的复制元件DUO也将被研究在翻译过程中与CAS的相互作用，允许关键的kl-H发夹结合CAS，阻止CAS与下游的3'CITE结合并干扰翻译因子的结合。目的3将采用诱变、SHAPE和EMSA来确定CAS是否与下游3' cite相互作用并破坏其稳定性，以及这是否会干扰翻译因子的结合和RdRp转录。Aim 4将研究DUO、kl-H和CAS之间的关系，以确定CAS如何被阻止结合其3'CITE。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project will benefit society by having college undergraduates, PhD level graduate students and postdoctoral researchers participate in research designed to address how viruses engage in one of their most important activities, synthesis of their required proteins. This research will be presented at scientific conferences, used in the teaching of introduction to biology for biology majors, and used to introduce undergraduates at the University of Maryland and Bowie State University to cutting edge procedures designed to study how the virus manipulates its genome in ways that were not previously thought to occur. In addition, this research adds a new dimension of understanding to a procedure (protein synthesis) that was thought to be mostly solved by exploring the possible regulation of a key aspect of virus biology: how they switch from being a template for making proteins to having that template replicated by changing how the template interacts with specific proteins. By studying three different viruses that all appear to use the same process to control how proteins are made, results should have a broad impact on virus research in general. Thus the results of this research should be applicable not only to plant viruses, but also animal and human viruses, including ones that cause serious disease in animals and plants. Recent results have led to the hypothesis that translation enhancers located in 3' UTRs of plant viruses need to be cleared of bound translation factors/ribosomes for replication of the template to proceed. PEMV contains three 3'cap-independent translation enhancers (3'CITEs): kl-TSS, PTE, and TSS, only two of which are used by the genomic RNA (kl-TSS/PTE) while all three are used by the subgenomic RNA. In Aim 1, SHAPE RNA structure probing and mutagenesis will be used to determine whether a sequence exclusive to the gRNA alters the structure of the TSS to keep it from functioning. In Aim 2, compensatory mutations and SHAPE will be used to examine a newly discovered element (the CAS) for its ability to block translation factor binding to downstream 3' CITEs in three viruses. The newly identified replication element DUO will also be investigated for interaction with CAS during translation, allowing the critical kl-H hairpin to bind CAS, keeping CAS from binding to the downstream 3'CITE and interfering with translation factor binding. Aim 3 will employ mutagenesis, SHAPE and EMSA to determine if CAS interacts with, and destabilizes its downstream 3'CITE, and whether this interferes with binding of translation factors and RdRp transcription. Aim 4 will investigate the relationship among DUO, kl-H and CAS to determine how CAS is blocked from binding its 3'CITE.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.