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嗜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嗜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，也表现出感染广谱人类癌症和转化细胞的能力。MYXV目前正被开发为一种溶瘤病毒治疗剂，用于治疗各种类型的癌症。我们将研究MYXV宿主范围蛋白M029的分子机制，该蛋白与多种宿主蛋白和介导宿主抗病毒反应的信号通路相互作用。M029是痘病毒E3家族的含有dsRNA结合结构域（dsRBD）的蛋白质的成员，但与牛痘病毒（VACV）E3版本不同，M029缺乏整个N末端“Z-DNA结合”结构域。我们已经报道了M029对于MYXV的体外和体内宿主和细胞向性，特别是在人细胞中是必需的。事实上，M029敲除MYXV具有我们所观察到的最明显的宿主范围缺陷，并且这种病毒不能在迄今为止测试的任何人类细胞中复制，这表明M029蛋白是旨在保护人类细胞免受病毒感染的细胞因子和途径的有效调节剂。因此，在人类细胞中解构M029靶标不仅对于MYXV作为治疗性溶瘤病毒的开发是重要的，而且作为研究已经进化以保护人类细胞免受病毒病原体侵害的先天和内在细胞限制因子的工具也是重要的。我们最近报道了M029蛋白结合并意外地激活关键的细胞RNA解旋酶RHA/DHX 9，以介导扩大的细胞向性，特别是在人类细胞中。我们还确定了几个额外的人类DDX/DHX RNA解旋酶作为M029的蛋白质相互作用伴侣，这表明RNA解旋酶可能在控制病毒宿主和细胞向性方面具有更广泛的作用。除了RNA解旋酶，M029还结合并抑制MYXV感染期间的蛋白激酶R（PKR）活化。因此，MYXV调节PKR和多个细胞RNA解旋酶通过一个单一的免疫调节蛋白，我们建议揭示新的分子机制，成功的MYXV复制在不同的细胞类型，特别是人类癌细胞，也是如何调节介导的发病机制，在其允许的兔宿主。根据我们的观察，我们建议调查以下内容：目的1。研究RHA/DHX 9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DHX 9和PKR功能的分子机制。此外，我们将研究M029对PKR和DHX 9的共调节如何也影响DHX 9介导的MYXV向性。我们将在重组MYXV中构建M029的突变体，这些突变体改变M029的这些相互作用和/或定位，并在培养细胞和病毒感染的兔中检查这些特定M029相互作用改变的后果。我们还将具体剖析DHX 9和PKR调节MYXV嗜性和人类癌细胞溶瘤的机制。目标2.阐明其他RNA解旋酶在MYXV复制和扩大细胞嗜性中的作用：使用所有人DDX/DHX RNA解旋酶和特定M029突变体的siRNA文库，我们鉴定了在不同细胞类型中最佳病毒基因表达和复制所需的几种其他RNA解旋酶。此外，我们已经确定了新的RNA解旋酶，相反，抑制MYXV在人类细胞中的复制。我们将研究这些细胞解旋酶如何介导MYXV的扩大宿主向性，特别是在人类癌细胞中。这些RNA解旋酶中的一些以dsRNA依赖性或非依赖性方式直接与M029相互作用。我们将使用M029突变体来研究这些相互作用在MYXV向性中的意义。我们将专门测试一些解旋酶在滴定中起作用的假设。 dsRNA水平，并确定特定的解旋酶成员是否以细胞类型特异性的方式作为痘病毒传感器起作用。