Innate Immune Regulation of Intracellular Pathways Involved in Filovirus Budding

埃博拉病毒（EBOV）和马尔堡病毒（MARV）是有包膜的负义RNA病毒，属于 丝状病毒科目前既没有疫苗也没有抗病毒药物来对抗这些危险的NIAID 一种病原体，引起人类高死亡率的出血综合征。我们的实验室专注于 丝状病毒“劫持”宿主蛋白质以调节出芽的机制，以及先天免疫系统如何 系统抵消这种相互作用以阻止病毒流出。 尽管EBOV VP 40在免疫中的作用是众所周知的， 促进病毒出芽的晚期阶段，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VP 40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，我们已经鉴定了宿主IQGAP 1作为EBOV VP 40的相互作用伴侣。 IQGAP 1是一个 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， 肌动蛋白丝组装以及丝状伪足和板状伪足的形成。 有趣的是，IQGAP 1拥有一个WW- 结构域能够与EBOV VP 40的PPxY型L结构域相互作用，我们的初步结果使用 siRNA证明IQGAP 1是VP 40病毒样颗粒（VLP）有效排出所必需的。基于 这些数据，我们将首先检验EBOV VP 40 L-结构域通过其功能依次招募IQGAP 1的假设。 WW-domain启动早期出芽事件，随后是宿主Nedd 4和/或Tsg 101促进晚期阶段 病毒-细胞分离（目的1）。重要的是，IQGAP 1被认为是IFN刺激的先天免疫缺陷的靶点。 免疫反应，提供了一个关键的第一道防线，抵御入侵的病原体。 事实上，干扰素 受激基因-15（ISG 15）是一种先天免疫应答基因，由于其 对包括DNA和RNA病毒在内的多种病原体具有广泛的抗病毒活性。 作为我们 初步结果支持IQGAP 1促进EBOV VLP出芽的假设，我们假设， ISG 15对宿主IQGAP 1的ISG化可能会破坏VP 40-IQGAP 1复合物，从而促进VP 40-IQGAP 1的表达。 随后出芽所需的功能性VP 40-Nedd 4和/或VP 40-Tsg 101相互作用降低。 这 宿主先天免疫防御和调节出芽的病毒编码因子之间的独特相互作用 将在目标2中探讨这一进程。 如果我们的假设是正确的，这些研究将：1）挑战目前的 病毒L-结构域基序仅在病毒出芽的后期步骤起作用的范例，2）鉴定新的、功能性的 EBOV VP 40与宿主蛋白/参与膜突起、丝状伪足的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，和3）揭示新的主机 先天免疫机制，调节高优先级的萌芽，新兴的病原体。

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.