Contributions of Ebola and Marburg virus VP30 and VP24 proteins to viral RNA synthesis, assembly and egress

关于埃博拉病毒和马尔堡病毒VP30和VP24蛋白贡献的RP02项目摘要/摘要 对病毒RNA的合成、组装和出口 马尔堡病毒(Marburg Virus，MARV)和埃博拉病毒(Ebola Virus，EBOV)是丝状病毒家族中新出现的致命性病毒。主要差距 关于丝状病毒-丝状病毒和丝状病毒-宿主的相互作用如何促进复制和 严重的疾病。造成这些知识局限性的原因是需要研究复制的丝状病毒 在生物安全4级(BSL4)围堵下。为了克服BSL4的限制，我们生产了复制 周期建模分析，称为转录复制能力病毒样颗粒(TrVLP)分析， 在BSL2总结EBOV和MARV复制周期中的每个主要步骤。这些工具可实现 在复制系统中对丝状病毒和宿主蛋白的有效分析。这些系统还 允许研究致命性突变或消除关键宿主因子等不是 兼容复制活病毒。通过这些检测，我们探索了Marv VP30(MVP30)的作用。 和Marv VP24(MVP24)，与它们的EBOV同源物相比，显示出不同的功能特性。 EBOV VP30是EBOV核蛋白(ENP)基因转录启动所必需的，受 带有PPxPxY基序的宿主蛋白。相反，据报道mVP30不是病毒转录所必需的 微型基因组分析，但它对病毒的生长是必不可少的。为什么它是必不可少的，目前还不清楚。EBOV VP24结合 Importinα蛋白阻断干扰素信号转导并参与核衣壳成熟 形成，调节病毒的RNA合成，是丝状病毒感染性的关键。相反，mVP24与 宿主蛋白Keap1，细胞抗氧化反应的调节者。我们的Marv trVLP数据显示mVP30 是有效生产活病毒所必需的，因为它需要MGP基因的转录，以及 破坏mVP24-Keap1相互作用的突变大大降低了传染性。鉴于这些发现，我们 将检验mVP30在MGP转录启动中起关键作用的假设；我们将表征 我们假设的二级结构赋予mVP30对MGP基因的依赖性；我们将评估 相互作用的宿主蛋白如何调节mVP30的功能。我们还将测试mVP24的假设 与Keap1的相互作用是病毒生长所必需的，并定义mVP24-Keap1的特定复制步骤 互动是必需的。最后，通过我们的trVLP分析，我们将确定其他丝状病毒- 研究项目1和3中确定的宿主相互作用会影响病毒的生长。总而言之，这些研究将 大大提高对丝状病毒复制和复制中起关键作用的相互作用的了解 发病机制。

RP02 Project Summary/Abstract for Contributions of Ebola and Marburg virus VP30 and VP24 proteins to viral RNA synthesis, assembly and egress Marburg virus (MARV) and Ebola virus (EBOV) are deadly emerging viruses of the filovirus family. Major gaps remain in our understanding as to how filoviral-filoviral and filoviral-host interactions promote replication and severe disease. Contributing to these limitations in knowledge is the requirement to study replicating filoviruses under biosafety level 4 (BSL4) containment. To overcome the BSL4 limitation, we have produced replication cycle modeling assays, called transcription-replication competent virus-like particle (trVLP) assays, that recapitulate each of the major steps in the EBOV and MARV replication cycles at BSL2. These tools enable the efficient analysis of filoviral and host proteins in the context of a replicating system. These systems also permit the study of manipulations such as lethal mutations or elimination of key host factors that are not compatible with replication of live virus. With these assays, we have explored the role of MARV VP30 (mVP30) and MARV VP24 (mVP24) that exhibit distinct functional properties compared to their EBOV homologues. EBOV VP30 is required for transcription initiation of the EBOV nucleoprotein (eNP) gene and is regulated by host proteins with PPxPxY motifs. In contrast, mVP30 is reported to not be required for viral transcription in minigenome assays, yet it is essential for virus growth. Why it is essential has been unclear. EBOV VP24 binds importin alpha proteins to block interferon signaling and also participates in the maturation of nucleocapsid formation, modulates viral RNA synthesis, and is critical for filoviral infectivity. In contrast, mVP24 interacts with host protein Keap1, a regulator of cellular antioxidant responses. Our MARV trVLP data suggests that mVP30 is essential for efficient production of viable virus, due to its requirement for transcription of the mGP gene, and that mutations disrupting mVP24-Keap1 interaction substantially decrease infectivity. Given these findings, we will test the hypothesis that mVP30 plays a critical role in mGP transcription initiation; we will characterize secondary structures that we hypothesize confer mVP30 dependence on the mGP gene; and we will evaluate how interacting host proteins modulate mVP30 function. We will also test the hypothesis that mVP24 interaction with Keap1 is required for viral growth and define specific replication steps for which mVP24-Keap1 interaction is required. Finally, with our trVLP assays, we will define the mechanisms by which other filovirus- host interactions identified in Research Projects 1 and 3 affect virus growth. Together, these studies will substantially enhance the understanding of interactions that play key roles in filovirus replication and pathogenesis.