Studies in Poxvirus Host Range Genes and Tropism

 描述(由申请人提供)：痘病毒宿主范围基因和嗜性项目的研究在本提案中，我们将揭示粘液瘤病毒(MYXV)宿主相互作用蛋白调节细胞和组织功能的基本机制，这些功能调节宿主范围和细胞嗜性。MYXV是一种兔特有的痘病毒，也显示出感染广泛范围的人类癌症和转化细胞的能力。MYXV目前正在作为一种溶瘤病毒疗法开发，用于治疗各种类型的癌症。我们将研究MYXV宿主范围蛋白M029使用的分子机制，它与多个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dsRNA结合结构域(DsRBD)的成员，但与痘苗病毒(VACV)E3版本不同的是，M029缺乏完整的N端“Z-DNA结合”结构域。我们已经报道，M029对MYXV的体外和体内宿主和细胞趋向性是必不可少的，特别是在人类细胞中。事实上，M029基因敲除的MYXV具有我们所观察到的最明显的宿主范围缺陷，并且该病毒不能在迄今测试的任何人类细胞中复制，这表明M029蛋白是一种强大的细胞因子和途径的调节器，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靶点不仅对于将MYXV发展成为一种治疗性溶瘤病毒很重要，而且还可以作为一种工具来研究进化出的保护人类细胞免受病毒病原体侵袭的先天和内在细胞限制因子。我们最近报道，M029蛋白结合并意外激活一个关键的细胞RNA解旋酶RHA/DHX9，以介导扩大的细胞趋向性，特别是在人类细胞中。我们还鉴定了另外几个人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嗜性方面可能具有更广泛的作用。在MYXV感染过程中，除RNA解旋酶外，M029还结合并抑制蛋白激酶R(PKR)的激活。因此，MYXV通过单一的免疫调节蛋白调节PKR和多个细胞RNA解旋酶，我们建议揭示新的分子机制，这些机制对于MYXV在不同类型的细胞中成功复制至关重要，特别是在人类癌细胞中，以及这种调节剂如何在其允许的兔宿主中介导发病。基于我们的观察，我们建议进行以下研究：目的1.研究RHA/DHX9和PKR在MYXV复制、发病机制和细胞趋向性中的作用：我们将研究M029调节DHX9和PKR功能的分子机制。此外，我们还将研究M029对PKR和DHX9的共同调节如何影响DHX9介导的MYXV的趋向性。我们将在重组MYXV中构建M029的突变体，以改变M029的这些相互作用和/或定位，并检测这些特定的M029相互作用改变在培养细胞和感染病毒的兔子中的后果。我们还将具体剖析DHX9和PKR调节人类癌细胞MYXV趋向性和溶瘤的机制。目的2.阐明其他RNA解旋酶在MYXV复制和扩大细胞趋向性中的作用：利用所有人DDX/DHX RNA解旋酶和特定的M029突变体的siRNA文库，我们鉴定了其他几种RNA解旋酶，它们是病毒基因在不同细胞类型中最佳表达和复制所必需的。此外，我们已经确定了新的RNA解旋酶，相反，它抑制了人类细胞中的MYXV复制。我们将研究这些细胞解旋酶如何介导MYXV的扩展宿主取向，特别是在人类癌细胞中。其中一些RNA解旋酶以dsRNA依赖或独立的方式与M029直接相互作用。我们将使用M029突变体来研究这些相互作用在MYXV趋向性中的意义。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定的方式作为痘病毒传感器发挥作用。

 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.