Mechanisms of Marburg virus gene expression

马尔堡病毒（MARV）属于丝状病毒科，在人类中具有高致病性。尽管被NIAID列为A类优先病原体，并且有可能导致大规模爆发，类似于最近的埃博拉病毒爆发，但对MARV的研究明显落后于其他非节段负义（NNS）RNA病毒。在这里，我们建议对MARV的转录和基因表达进行深入分析。剖析MARV基因表达的机制不仅有助于抗病毒药物的靶向开发，还将揭示NNS RNA病毒之间的统一范式和区别。丝状病毒基因组由病毒编码的RNA依赖性RNA聚合酶复合体转录，该复合体能够产生加帽和聚腺苷酸化的mRNA。这一过程发生在细胞质中，靠近核糖体和细胞RNA结合蛋白。该项目将研究MARV基因表达的三个不同阶段。在目的1中，我们将阐明在MARV启动子转录起始的机制，并调查在这一过程中的聚合酶的结构特征的作用。值得注意的是，MARV启动子序列有一些不寻常的功能，我们打算探索这些功能的相关性。在目标2中，我们将确定在每个MARV mRNA的5 ′端形成的保守发夹环的功能。我们将探讨这些结构对转录，RNA稳定性，运输和翻译的影响。在目标3中，我们将专注于mRNA聚腺苷酸化和释放。对于任何NNS RNA病毒，mRNA释放的机制都没有很好的理解，在这个目标中获得的结果将有助于拓宽我们对NNS RNA病毒转录策略的理解。该提案汇集了研究NNS RNA聚合酶，MARV分子生物学和mRNA-蛋白质相互作用的专业知识。研究团队共同建立了一套独特的工具来实现这一提议的目标，包括MARV体外聚合酶测定、各种MARV反向遗传学系统和高度创新的单分子mRNA-蛋白质结合测定。这些研究将为MARV感染的一个关键方面提供新的线索，并增强我们对NNS RNA病毒生物学的理解。

Marburg virus (MARV) belongs to the filovirus family and is highly pathogenic in humans. Despite being classified as Category A Priority Pathogen by NIAID, and its potential to cause large-scale outbreaks, similar to the recent Ebola virus outbreak, research on MARV lags significantly behind that on other non-segmented negative sense (NNS) RNA viruses. Here, we propose to perform in-depth analyses of MARV transcription and gene expression. Dissecting the mechanisms of MARV gene expression will not only be instrumental for the targeted development of antiviral drugs, it will also reveal unifying paradigms and distinctions between the NNS RNA viruses. The filovirus genome is transcribed by a virally encoded RNA-dependent RNA polymerase complex, which is capable of generating capped and polyadenylated mRNAs. This process occurs in the cell cytoplasm, close to ribosomes and cellular RNA binding proteins. This project will examine three different stages of MARV gene expression. In Aim 1, we will elucidate the mechanism of transcription initiation at the MARV promoter and investigate the role of structural features of the polymerase in this process. Notably, the MARV promoter sequence has some unusual features, and we intend to explore the functional relevance of these characteristics. In Aim 2, we will determine the function of conserved hairpin loops that are formed at the 5´ end of each MARV mRNA. We will explore the effect of these structures on transcription, RNA stability, trafficking and translation. In Aim 3, we will focus on mRNA polyadenylation and release. The mechanism of mRNA release is not well understood for any NNS RNA virus, and the results obtained in this aim will help to broaden our understanding of NNS RNA virus transcription strategies. This proposal brings together expertise in studying NNS RNA polymerases, MARV molecular biology, and mRNA-protein interactions. Together, the research team has established a unique tool set to achieve the goals of this proposal, including a MARV in vitro polymerase assay, various MARV reverse genetics systems, and highly innovative single molecule mRNA-protein binding assays. These studies will shine new light on a crucial aspect of MARV infection and enhance our understanding of NNS RNA virus biology.