Modulation of Innate Immune Responses by Cytomegalovirus

描述(申请人提供)：干扰素(干扰素)和干扰素刺激基因产物(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ISGs的诱导或功能，才能成功感染它们各自的宿主。人类最常见的病原体之一是人类巨细胞病毒(HCMV)，但目前尚不清楚HCMV是如何激活和调节干扰素应答的。因此，这项应用的目标是识别和表征针对HCMV的这种先天免疫反应的细胞激活剂和病毒调节器。我们已经证明，干扰素调节因子3(IRF3)的激活在巨细胞病毒诱导的干扰素和胰岛素样免疫球蛋白的诱导中是必不可少的。然而，我们也观察到非人灵长类病毒恒河猴CMV(RhCMV)的病毒粒子蛋白可以阻止HCMV激活IRF3。RhCMV被膜的两种蛋白pp65a和pp71抑制依赖于IRF3的ISG诱导。出乎意料的是，HCMV的pp71(UL82)也抑制了依赖IRF3的ISG转录。有趣的是，稳定转染pp72(UL82-HF)的人成纤维细胞对HCMV激活的IRF3完全无效，但不能被水泡性口炎病毒或PolyIC激活。此外，双链干扰素刺激DNA(ISD)不能激活UL82-HF中的IRF3。依赖ISD的IRF3激活是最近被描述的一种新的先天性免疫反应途径，它不依赖于Toll样受体或dsRNA传感器RIG-I和MDA-5。因此，我们推测HCMV和ISD共有一个共同的IRF3激活信号转导途径，该信号转导途径受CMV被膜蛋白的调控。为了验证这一假设，我们计划确定HCMV和/或依赖ISD的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。我们将通过确定RhCMV和HCMV被蛋白靶向的信号转导级联中的步骤来进一步确定病毒IRF3抑制的机制。最后，我们将描述病毒被膜蛋白在病毒感染过程中调节IRF3激活的作用。最终，这些体外结果将使我们能够在一种新的巨细胞病毒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.