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

埃博拉病毒和马尔堡病毒 VP30 和 VP24 蛋白对病毒 RNA 合成、组装和流出的贡献

• 批准号: 10555056
• 负责人: Christopher F Basler
• 金额: $ 43.64万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-07 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10555056
• 关键词: Address; Affect; Antioxidants; Binding; Biological Assay; Biophysics; CRISPR/Cas technology; Cells; Containment; Cytoprotection; Data; Dependence; Disease; Ebola virus; Exhibits; Family; Filovirus; Genes; Genetic Transcription; Goals; Growth; Homologous Gene; Infection; Integration Host Factors; Interferons; Knock-out; Knowledge; Length; MGP gene; Marburgvirus; Mediating; Modeling; Modification; Molecular; Mutation; Nuclear Family; Nuclear Import; Nucleocapsid; Nucleoproteins; Outcome; Pathogenesis; Pathway interactions; Peptides; Play; Production; Property; Protein Truncation; Proteins; RNA; RNA Processing; RNA analysis; RNA chemical synthesis; RNA-Protein Interaction; Recombinants; Regulation; Reporting; Research Project Grants; Role; Signal Transduction; Small Interfering RNA; Structure; System; TRIP10 gene; Testing; Transcription Initiation; Transcription Initiation Site; Transcriptional Regulation; Transfection; Viral; Viral Structural Proteins; Virion; Virus; Virus Diseases; Virus Replication; Virus-like particle; Work; alpha Karyopherins; member; mutant; novel; overexpression; recombinant virus; response; stem; success; tool; transcription factor; viral RNA

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.

RP 02埃博拉和马尔堡病毒VP 30和VP 24蛋白贡献的项目总结/摘要 病毒RNA的合成、装配和排出 马尔堡病毒（MARV）和埃博拉病毒（EBOV）是丝状病毒家族的致命的新兴病毒。重大差距 我们仍然理解丝状病毒-丝状病毒和丝状病毒-宿主相互作用如何促进复制， 严重的疾病。导致这些知识局限性的原因是需要研究复制型丝状病毒 生物安全等级4（BSL 4）。为了克服BSL 4的限制，我们已经生产了复制 循环建模测定，称为转录-复制能力病毒样颗粒（trVLP）测定， 概括了BSL 2的EBOV和MARV复制周期中的每个主要步骤。这些工具可以 在复制系统中对丝状病毒和宿主蛋白进行有效分析。这些系统还 允许研究操纵，如致命突变或消除关键的宿主因子， 与活病毒的复制相容。通过这些检测，我们探索了MARV VP 30（mVP 30）在 和MARV VP 24（mVP 24），其与它们的EBOV同源物相比表现出不同的功能特性。 EBOV VP 30是EBOV核蛋白（eNP）基因转录起始所必需的，并受 具有PPxPxY基序的宿主蛋白。相比之下，据报道mVP 30在大肠杆菌中不是病毒转录所需的。 微型基因组测定，但它是必不可少的病毒生长。为什么它是必不可少的一直不清楚。EBOV VP 24结合 输入素α蛋白阻断干扰素信号传导，也参与核衣壳的成熟 它是丝状病毒的重要组成部分，调节病毒RNA的合成，并且对丝状病毒的感染性至关重要。相反，mVP 24与 宿主蛋白Keap 1，细胞抗氧化反应的调节剂。我们的MARV trVLP数据表明，mVP 30 由于其需要mGP基因的转录，因此对于有效生产活病毒是必不可少的， 破坏mVP 24-Keap 1相互作用的突变显著降低了感染性。鉴于这些发现，我们 将检验mVP 30在mGP转录起始中起关键作用的假设;我们将表征 二级结构，我们假设赋予mVP 30依赖于mGP基因，我们将评估 相互作用的宿主蛋白如何调节mVP 30功能。我们还将检验mVP 24 与Keap 1的相互作用是病毒生长所必需的，并定义了mVP 24-Keap 1 需要互动。最后，通过我们的trVLP检测，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影响病毒生长。这些研究将 大大增强了对在丝状病毒复制中发挥关键作用的相互作用的理解， 发病机制