Innate Immune Regulation of Intracellular Pathways Involved in Filovirus Budding

埃博拉病毒(EBOV)和马尔堡病毒(Marburg)属于包膜负义RNA病毒家族 丝状病毒科。目前还没有疫苗或抗病毒药物来对抗这些危险的NIAID类别 一种病原体，可导致人类高死亡率的出血性综合征。我们的实验室专注于 关于丝状病毒“劫持”宿主蛋白以调节发芽的机制，以及天然免疫如何 系统会抵消这种相互作用，以阻止病毒外泄。尽管EBOV VP40在 促进病毒萌发的后期，其对早期萌发阶段(例如细胞骨架)的潜在贡献 重塑和最初的芽突起)几乎是未知的。在这里，我们将探索一台出乎意料的VP40主机 可能促进病毒萌发早期阶段的相互作用及其先天免疫的潜在调节 系统。值得注意的是，我们已经将主机IQGAP1确定为EBOV VP40的交互伙伴。IQGAP1是一个 多结构域蛋白，协调形成与调节细胞运动有关的蛋白质复合体， 肌动蛋白细丝组装，丝足和片脂的形成。有趣的是，IQGAP1拥有一个WW- 能够与EBOVVP40的PPxY型L结构域相互作用的结构域，以及我们使用 SiRNA表明，IQGAP1是VP40病毒样颗粒(VLP)有效出口所必需的。基于 这些数据，我们将首先检验EBOVVP40 L结构域通过ITS顺序招募IQGAP1的假设 WW-DOMAIN发起早期萌芽活动，随后是主机Nedd4和/或Tsg101以促进后期 病毒-细胞分离(目标1)。重要的是，IQGAP1被认为是干扰素刺激的先天性 免疫反应，提供抵御入侵病原体的关键第一道防线。的确，干扰素 刺激基因-15(ISG15)是一种先天免疫反应基因，因其特有的功能而备受关注。 对包括DNA和RNA病毒在内的大量病原体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发芽的假设，我们假设 ISG15对宿主IQGAP1的ISG基化可能破坏VP40-IQGAP1复合体，从而促进 发芽所需的VP40-Nedd4和/或VP40-Tsg101相互作用随后减少。这 宿主先天免疫防御和病毒编码因子之间独特的相互作用调节萌发 如果我们的假设是正确的，这些研究将：1)挑战当前的 病毒L结构域仅在病毒萌发的后期发挥作用的范式，2)识别新的、有功能的 EBOV VP40与膜突起、丝状伪足等宿主蛋白/途径的相互作用 形成和/或肌动蛋白细胞骨架重塑，有助于早期萌发事件，以及3)揭示新的宿主 调节高优先级新兴病原体萌发的先天免疫机制。

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.