Modulation of Innate Immune Responses by Cytomegalovirus

描述（由申请人提供）：干扰素（IFN）和IFN刺激的基因产物（ISGs）是对病毒感染的先天免疫反应的重要组成部分。相反，病毒需要限制isg的诱导或功能才能成功感染各自的宿主。最普遍的人类病原体之一是乙型疱疹病毒人巨细胞病毒（HCMV），但HCMV如何激活和调节IFN反应尚不清楚。因此，本应用程序的目标是识别和表征HCMV先天免疫反应的细胞激活剂和病毒调节剂。我们已经证明干扰素调节因子3 （IRF3）的激活对于hcmv诱导的IFN和ISG诱导至关重要。然而，我们也观察到非人灵长类病毒恒河巨细胞病毒（RhCMV）的病毒粒子蛋白可以阻止IRF3被HCMV激活。RhCMV被膜的两个蛋白pp65a和pp71抑制irf3依赖性ISG诱导。出乎意料的是，HCMV的pp71 （UL82）也抑制了irf3依赖性ISG的转录。有趣的是，稳定转染pp72 （UL82-HF）的人成纤维细胞对HCMV激活IRF3完全不耐受，但对水疱性口炎病毒或poly-IC不耐受。此外，在UL82-HF中，双链干扰素刺激DNA （ISD）未能激活IRF3。isd依赖性IRF3激活是最近被描述的一种新的先天免疫反应途径，它不依赖于toll样受体或dsrna传感器RIG-I和MDA-5。因此，我们假设HCMV和ISD具有共同的irf3激活信号转导途径，该途径由CMV被膜蛋白调节。为了验证这一假设，我们计划鉴定HCMV和/或ISD依赖性IRF3激活所需的宿主细胞因子。这将在针对大约17,000个已知和未知基因的小干扰rna的高通量筛选中实现。我们将通过识别RhCMV和HCMV被膜蛋白靶向的信号转导级联中的步骤，进一步确定病毒irf3抑制的机制。最后，我们将描述病毒被膜蛋白在病毒感染期间调节IRF3激活中的作用。最终，这些体外结果将使我们能够在新兴的HCMV动物模型中测试调节先天免疫反应的重要性。

DESCRIPTION (provided by applicant): Interferon (IFN) and IFN-stimulated gene products (ISGs) are essential components of the innate immune response to viral infection. Conversely, viruses need to limit the induction or function of ISGs for successful infection of their respective host. One of the most highly prevalent human pathogens is the beta-herpesvirus human cytomegalovirus (HCMV), yet it is not clear how HCMV activates and modulates the IFN response. The goal of this application is therefore to identify and characterize cellular activators and viral modulators of this innate immune response to HCMV. We have shown that the activation of interferon-regulatory factor 3 (IRF3) is essential for HCMV-induced IFN and ISG induction. However, we also observed that virion proteins of the non-human primate virus rhesus CMV (RhCMV) prevent IRF3 activation by HCMV. Two proteins of the RhCMV tegument, pp65a and pp71 inhibit IRF3-dependent ISG induction. Unexpectedly, pp71 (UL82) of HCMV also inhibited IRF3-dependent ISG transcription. Interestingly, human fibroblasts stably transfected with pp72 (UL82-HF) were completely refractory to IRF3 activation by HCMV, but not by Vesicular Stomatitis Virus or poly-IC. In addition, double stranded, interferon-stimulatory DNA (ISD) failed to activate IRF3 in UL82-HF. ISD-dependent IRF3 activation is a recently described novel innate immune response pathway that is independent of toll-like receptors or the dsRNA-sensors RIG-I and MDA-5. Therefore, we hypothesize that HCMV and ISD share a common IRF3-activating signal transduction pathway that is modulated by CMV tegument proteins. To test this hypothesis we plan to identify host cell factors required for HCMV and/or ISD- dependent IRF3 activation. This will be achieved in a high-throughput screen of small intefering RNAs targeting approximately 17,000 known and unknown genes. We will further identify the mechanism of viral IRF3-inhibtion by identifying the step within the signal transduction cascade that is targeted by tegument proteins of RhCMV and HCMV. Finally, we will characterize the role of viral tegument proteins in modulating IRF3 activation during viral infection. Ultimately, these in vitro results will allow us to test the importance of modulating the innate immune response in an emerging animal model for HCMV.