Studies in Poxvirus Host Range Genes and Tropism

痘病毒宿主范围基因和趋向性研究

• 批准号: 9384142
• 负责人: Grant McFadden
• 金额: $ 38.63万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-01 至 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9384142
• 关键词: Address; Affect; Alpha Cell; Antiviral Response; Area; Binding; Binding Proteins; Cells; Cellular Tropism; Cultured Cells; DNA Binding Domain; DNA Viruses; Defect; Defense Mechanisms; Development; Double-Stranded RNA; Ensure; Exhibits; Family; Gene Expression; Genes; Genome; Goals; Human; In Vitro; Knock-out; Knowledge; Lagomorpha; Leporipoxvirus; Libraries; Malignant Neoplasms; Mediating; Modeling; Molecular; Myxoma virus; N-terminal; Nature; Oncolytic; Oncolytic viruses; Oryctolagus cuniculus; Pathogenesis; Pathway interactions; Play; Poxviridae; Proteins; RNA Helicase; RNA helicase A; Recombinants; Regulation; Reporting; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Testing; Therapeutic; Tissues; Tropism; Vaccinia virus; Viral; Viral Genes; Viral Proteins; Virotherapy; Virus; Virus Diseases; Virus Replication; Z-Form DNA; Zoonoses; base; cancer cell; cell transformation; cell type; design; helicase; immunoregulation; in vivo; member; metaplastic cell transformation; mutant; novel; oncolysis; oncolytic virotherapy; pathogen; permissiveness; protein kinase R; public health relevance; sensor; tool; virus host interaction; virus tropism

 DESCRIPTION (provided by applicant): Studies in poxvirus host range genes and tropism Project Summary In this proposal we will uncover the fundamental mechanisms by which Myxoma virus (MYXV) host interactive proteins modulate cellular and tissue functions that mediate host range and cell tropism. MYXV is a rabbit specific poxvirus that also exhibits the capacity to infect a wide spectrum of human cancer and transformed cells. MYXV is currently being developed as an oncolytic virotherapeutic to treat various classes of cancer. We will examine the molecular mechanisms used by MYXV host range protein, M029, which interacts with multiple host proteins and signaling pathways that mediate host anti-viral responses. M029 is a member of the poxvirus E3 family of dsRNA Binding Domain (dsRBD) containing proteins, but unlike the vaccinia virus (VACV) E3 version, M029 lacks the entire N terminal "Z-DNA binding" domain. We have reported that M029 is essential for both in vitro and in vivo host and cellular tropism of MYXV, particularly in human cells. Indeed, the M029- knockout MYXV has the most pronounced host range defect we have ever observed, and this virus cannot replicate in any human cell tested to date, indicating that the M029 protein is a potent modulator of cellular factors and pathways designed to protect human cells from virus infections. Thus, deconstructing M029 targets in human cells is important not only for the development of MYXV as a therapeutic oncolytic virus, but also as a tool to investigate innate and intrinsic cellular restriction factors that have evolved to protect human cells from viral pathogens. We have recently reported that the M029 protein binds and unexpectedly activates a key cellular RNA helicase, RHA/DHX9, to mediate expanded cellular tropism, particularly in human cells. We have also identified several additional human DDX/DHX RNA helicases as protein-interacting partners of M029, suggesting that RNA helicases may have a much broader role in governing viral host and cellular tropism. In addition to RNA helicases, M029 also binds and inhibits protein kinase R (PKR) activation during MYXV infection. MYXV thus regulates both PKR and multiple cellular RNA helicases via a single immunomodulatory protein, and we propose to uncover novel molecular mechanisms that are crucial for successful MYXV replication in diverse cell types, particularly human cancer cells, and also how this modulator mediates pathogenesis in its permissive rabbit host. Based on our observations we propose to investigate the followings: Aim 1. Investigate the role of RHA/DHX9 and PKR in MYXV replication, pathogenesis and cellular tropism: We will examine the molecular mechanisms by which M029 modulates DHX9 and PKR functions. In addition, we will investigate how the co-regulation of PKR and DHX9 by M029 also affects DHX9-mediated tropism of MYXV. We will construct mutants of M029 within recombinant MYXV that alter these interactions and/or localization of M029, and examine the consequences of these specific M029-interactive alterations in both cultured cells and in virus-infected rabbits. We will also specifically dissect the mechanisms by which DHX9 and PKR regulate MYXV tropism and oncolysis of human cancer cells. Aim 2. Elucidate the role of other RNA helicases in MYXV replication and expanding cellular tropism: Using an siRNA library for all the human DDX/DHX RNA helicases, and specific M029 mutants, we identified several other RNA helicases that are required for optimum viral gene expression and replication in diverse cell types. Additionally, we have identified new RNA helicases that, in contrast, inhibit MYXV replication in human cells. We will study how these cellular helicases mediate the expanded host tropism of MYXV, particularly in human cancer cells. Some of these RNA helicases interact directly with M029, either in a dsRNA dependent or independent manner. We will use M029 mutants to investigate the significance of these interactions in MYXV tropism. We will specifically test the hypothesis that some of the helicases play role in titrating dsRNA levels and determine whether specific helicase members function as poxvirus sensors in a cell type specific manner.

 描述（由申请人提供）：痘病毒宿主范围基因和向性的研究 项目摘要 在本提案中，我们将揭示粘液瘤病毒 (MYXV) 宿主相互作用蛋白调节介导宿主范围和细胞向性的细胞和组织功能的基本机制。 MYXV 是一种兔特异性痘病毒，也具有感染多种人类癌症和转化细胞的能力，目前正在开发一种溶瘤病毒治疗剂。我们将研究 MYXV 宿主范围蛋白 M029 所使用的分子机制，它与介导宿主抗病毒反应的多种宿主蛋白​​和信号通路相互作用。M029 是 dsRNA 结合的痘病毒 E3 家族的成员。含有蛋白质的结构域（dsRBD），但与痘苗病毒（VACV）E3版本不同，M029缺乏整个N末端“Z-DNA结合”结构域。 M029 对于 MYXV 的体外和体内宿主和细胞趋向性至关重要，尤其是在人类细胞中。事实上，M029 敲除的 MYXV 具有我们观察到的最明显的宿主范围缺陷，并且该病毒无法在任何人类细胞中复制。迄今为止的测试表明，M029 蛋白是一种有效的细胞因子和途径调节剂，旨在保护人类细胞免受病毒感染，因此，解构人类细胞中的 M029 靶标不仅对于病毒的发展很重要。 MYXV 作为一种治疗性溶瘤病毒，也是研究先天性和内在细胞限制因子的工具，这些限制因子已经进化以保护人类细胞免受病毒病原体的侵害。我们最近报道，M029 蛋白结合并意外地激活了关键的细胞 RNA 解旋酶 RHA。 /DHX9，介导扩大的细胞向性，特别是在人类细胞中。我们还鉴定了几种其他人 DDX/DHX RNA 解旋酶作为 M029 的蛋白质相互作用伙伴，这表明RNA 解旋酶在控制病毒宿主和细胞趋向性方面可能具有更广泛的作用，除了 RNA 解旋酶外，M029 还可以在 MYXV 感染期间结合并抑制蛋白激酶 R (PKR) 激活，从而通过 a 调节 PKR 和多种细胞 RNA 解旋酶。单一免疫调节蛋白，我们建议揭示对于 MYXV 在不同细胞类型（特别是人类癌细胞）中成功复制至关重要的新分子机制，以及该调节剂如何介导根据我们的观察，我们建议研究以下内容： 目标 1. 研究 RHA/DHX9 和 PKR 在 MYXV 复制、发病机制和细胞向性中的作用：我们将研究 M029 调节 DHX9 的分子机制。此外，我们将研究 M029 对 PKR 和 DHX9 的共同调节如何影响 DHX9 介导的。我们将在重组 MYXV 中构建 M029 突变体，以改变 M029 的这些相互作用和/或定位，并检查这些特定的 M029 相互作用改变在培养细胞和病毒感染的兔子中的后果。剖析 DHX9 和 PKR 调节 MYXV 趋向性和人类癌细胞溶瘤作用的机制。阐明其他 RNA 解旋酶在 MYXV 复制和扩大细胞向性中的作用：使用所有人类 DDX/DHX RNA 解旋酶和特定 M029 突变体的 siRNA 文库，我们鉴定了最佳病毒基因表达和复制所需的几种其他 RNA 解旋酶此外，我们还发现了新的 RNA 解旋酶，相反，我们将研究这些细胞解旋酶如何介导。扩大了 MYXV 的宿主向性，特别是在人类癌细胞中，其中一些 RNA 解旋酶以 dsRNA 依赖性或独立方式直接与 M029 相互作用。特别检验一些解旋酶在滴定中发挥作用的假设 dsRNA 水平并确定特定解旋酶成员是否以细胞类型特异性方式充当痘病毒传感器。