Innate Immune Regulation of Intracellular Pathways Involved in Filovirus Budding

丝状病毒出芽涉及的细胞内途径的先天免疫调节

基本信息

批准号：
8635498
负责人：
RONALD N HARTY
金额：
$ 24万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-12-01 至 2015-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8635498
关键词：
Actins Antiviral Agents Attention Biological Assay Bioterrorism Categories Cell Separation Cell membrane Cells Co-Immunoprecipitations Complex DNA Viruses Data Defense Mechanisms Development Disease Event Family Filopodia Filoviridae Filovirus Fluorescence Fluorescence Resonance Energy Transfer Funding Mechanisms Future Genes Host Defense Human Image Imaging Techniques Immune Immune Response Genes Immune response Immune system In Vitro Infection Interferons Invaded Laboratories Life Maps Mediating Membrane Microfilaments Microscopy Mono-S Morphology National Institute of Allergy and Infectious Disease Nature Pathway interactions Peptide Hydrolases Process Production Proline Proteins RNA Viruses Recruitment Activity Regulation Reporting Role Site Small Interfering RNA Staging Syndrome System Testing Ubiquitination Vaccines Vacuolar Protein Sorting Viral Viral Matrix Proteins Virion Virus Virus Diseases Virus-like particle base cell motility combat fighting innovation mortality mutant novel novel strategies novel therapeutics pathogen polymerization protein complex public health relevance small hairpin RNA transmission process viral RNA

项目摘要

Ebola (EBOV) and Marburg (MARV) are enveloped, negative-sense RNA viruses belonging to the family Filoviridae. Neither vaccines nor antivirals are currently available to combat these dangerous NIAID Category A pathogens, which cause hemorrhagic syndromes with high mortality rates in humans. Our laboratory focuses on the mechanisms by which filoviruses "hijack" host proteins to regulate budding, and how the innate immune system counteracts such interactions to block virus egress. Despite the well known role of EBOV VP40 in promoting late stages of virus budding, its potential contributions to early budding stages (e.g. cytoskeletal remodeling and initial bud protrusion) are virtually unknown. Here, we will explore an unanticipated VP40-host interaction that may promote early stages of viral budding and its potential regulation by the innate immune system. Notably, we have identified host IQGAP1 as an interacting partner for EBOV VP40. IQGAP1 is a multidomain protein that orchestrates the formation of protein complexes involved in regulating cell motility, actin filament assembly, and filopodia and lamellipodia formation. Intriguingly, IQGAP1 possesses a WW- domain capable of interacting with a PPxY type L-domain of EBOV VP40, and our preliminary results using siRNA demonstrate that IQGAP1 is required for efficient egress of VP40 virus-like particles (VLPs). Based on these data, we will first test the hypothesis that EBOV VP40 L-domains sequentially recruit IQGAP1 via its WW-domain to initiate early budding events, followed by host Nedd4 and/or Tsg101 to facilitate late stage virus-cell separation (Aim 1). Importantly, IQGAP1 has been implicated as a target of the IFN-stimulated innate immune response that provides a critical first line of defense against invading pathogens. Indeed, interferon stimulated gene-15 (ISG15) is an innate immune response gene which has garnered recent attention due to its broad-range of antiviral activity against a plethora of pathogens including DNA and RNA viruses. As our preliminary results support the hypothesis that IQGAP1 promotes EBOV VLP budding, we hypothesize that ISGylation of host IQGAP1 by ISG15 may disrupt VP40-IQGAP1 complexes, thereby contributing to the subsequent decrease in functional VP40-Nedd4 and/or VP40-Tsg101 interactions required for budding. This unique interplay between host innate immune defenses and viral encoded factors that regulate the budding process will be explored in Aim 2. If our hypotheses are correct, these studies will: 1) challenge the current paradigm that viral L-domain motifs function only at late steps of virus budding, 2) identify novel, functional interactions between EBOV VP40 and host proteins/pathways involved in membrane protrusion, filopodia formation, and/or actin cytoskeletal remodeling that contribute to early budding events, and 3) reveal new host innate immune mechanisms that regulate budding of high priority, emerging pathogens.

埃博拉病毒 (EBOV) 和马尔堡病毒 (MARV) 是属于该科的有包膜负义 RNA 病毒丝状病毒科。目前还没有疫苗和抗病毒药物可以对抗这些危险的 NIAID 类别一种病原体，可引起人类高死亡率的出血综合征。我们的实验室重点关于丝状病毒“劫持”宿主蛋白来调节出芽的机制，以及先天免疫如何系统抵消这种相互作用以阻止病毒出口。尽管 EBOV VP40 在促进病毒出芽的后期阶段，其对早期出芽阶段的潜在贡献（例如细胞骨架重塑和初始芽突出）实际上是未知的。在这里，我们将探索一个意想不到的VP40主机可能促进病毒出芽早期阶段的相互作用及其通过先天免疫的潜在调节系统。值得注意的是，我们已经确定宿主 IQGAP1 是 EBOV VP40 的相互作用伙伴。 IQGAP1 是协调参与调节细胞运动的蛋白质复合物形成的多结构域蛋白质，肌动蛋白丝组装，以及丝状伪足和片状伪足的形成。有趣的是，IQGAP1 拥有 WW- 能够与 EBOV VP40 的 PPxY 型 L 结构域相互作用的结构域，以及我们使用的初步结果 siRNA 证明 IQGAP1 是 VP40 病毒样颗粒 (VLP) 有效流出所必需的。基于根据这些数据，我们将首先检验以下假设：EBOV VP40 L 结构域通过其序列依次招募 IQGAP1 WW 域启动早期萌芽事件，随后宿主 Nedd4 和/或 Tsg101 促进后期阶段病毒-细胞分离（目标 1）。重要的是，IQGAP1 被认为是 IFN 刺激的先天性细胞因子的靶标。免疫反应提供了抵御病原体入侵的关键第一道防线。确实，干扰素刺激基因 15 (ISG15) 是一种先天性免疫反应基因，由于其对多种病原体（包括 DNA 和 RNA 病毒）具有广泛的抗病毒活性。作为我们的初步结果支持 IQGAP1 促进 EBOV VLP 出芽的假设，我们假设 ISG15 对宿主 IQGAP1 的 ISGylation 可能会破坏 VP40-IQGAP1 复合物，从而有助于随后出芽所需的功能性 VP40-Nedd4 和/或 VP40-Tsg101 相互作用减少。这宿主先天免疫防御和调节出芽的病毒编码因子之间独特的相互作用该过程将在目标 2 中进行探讨。如果我们的假设正确，这些研究将：1）挑战当前的病毒 L 结构域基序仅在病毒出芽的后期发挥作用的范式，2) 识别新颖的、功能性的 EBOV VP40 与参与膜突出、丝状伪足的宿主蛋白/途径之间的相互作用形成和/或肌动蛋白细胞骨架重塑，有助于早期出芽事件，3) 揭示新宿主调节高优先级新兴病原体萌芽的先天免疫机制。