Elucidating the Critical Role of Low-Density Lipoprotein Receptor Class A Domain Containing 3 in Venezuelan Equine Encephalitis Virus Neuron Infection and Pathogenesis

阐明含 3 的低密度脂蛋白 A 类受体结构域在委内瑞拉马脑炎病毒神经元感染和发病机制中的关键作用

• 批准号: 10728883
• 负责人: Natasha Monir Kafai
• 金额: $ 5.27万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10728883
• 关键词: Affect; Affinity; Alphavirus; Americas; Antiviral Therapy; Binding; Biological Assay; Bioterrorism; Birds; Blood - brain barrier anatomy; Body Weight decreased; Brain; Cell Culture Techniques; Cells; Cellular Tropism; Central Nervous System; Cessation of life; Chimeric Proteins; Clinical; Confocal Microscopy; Cryoelectron Microscopy; Culicidae; Data; Development; Disease; Encephalitis; Engineering; Enterobacteria phage P1 Cre recombinase; Epidemic; Equus caballus; Family; Fc domain; Goals; Histopathology; Hour; Human; Immune; Immunoglobulin G; In Vitro; Infection; Inflammation; Inflammatory Response; Invaded; Laboratories; Laboratory Personnel; Life; Link; Lipoprotein Receptor; Low Density Lipoprotein Receptor; Lymphoid Tissue; Mammals; Measures; Mediating; Modeling; Molecular; Mus; Mutagenesis; Mutate; Mutation; Myeloid Cells; Neuroglia; Neurologic; Neurons; Neurotropism; Organ; Pathogenesis; Pathology; Periodicals; Peripheral; Population; Publishing; Quantitative Reverse Transcriptase PCR; RNA Viruses; Reporting; Research; Risk; Role; Route; Serum; Site; Specificity; Stromal Cells; Structure; Testing; Therapeutic; Time; Tissues; Togaviridae; Treatment Efficacy; Tropism; Venezuelan Equine Encephalitis Virus; Viral; Viral Load result; Viral Pathogenesis; Viremia; Virus; Virus Diseases; aerosolized; antiviral drug development; cell type; cellular targeting; clinical development; decoy receptor 3; immune cell infiltrate; improved; in vivo; mouse model; neuroepithelium; novel; prevent; receptor; scavenger receptor; subcutaneous; transcytosis; transmission process; vector mosquito; viral RNA; virus tropism; weapons

PROJECT SUMMARY Alphaviruses are enveloped positive-stranded RNA viruses of the Togaviridae family whose natural endemic cycles occur between mosquito vectors and nonhuman mammal or avian species. Venezuelan equine encephalitis virus (VEEV) is an encephalitic alphavirus responsible for periodic epidemics in equine and human populations across the Americas. Its potential for use as an aerosolized bioterrorism weapon and the demonstrated risk it poses in research settings highlight the need for countermeasures. VEEV pathogenesis is characterized by central nervous system (CNS) involvement and damage to lymphoid tissues. Our laboratory published the discovery of a novel receptor for VEEV, low-density lipoprotein receptor class A domain containing 3 (LDLRAD3), required for infection and VEEV pathogenesis in vivo. We have demonstrated that a soluble fusion protein with LDLRAD3 linked to an IgG Fc domain (LDLRAD3-Fc) protects mice from lethal infection. Though this shows that identification of an entry receptor can become a target for countermeasures against VEEV, the role of LDLRAD3 in controlling VEEV pathogenesis is still not understood. The first Aim of my proposal will explore LDLRAD3-mediated tropism and neuroinvasion of VEEV. I hypothesize that LDLRAD3 is required for VEEV infection of myeloid cells in peripheral organs and neurons in the central nervous system, which contribute to the development of inflammation and clinical disease. This hypothesis will be tested using in vivo infection studies that analyze viral RNA levels, histopathology, and virus tropism during time courses of infection in Ldlrad3-/- mice. Cell-type specificity of LDLRAD3-mediated VEEV infection will be explored using mixed primary neuron cultures from Ldlrad3-/- mice and tissue-specific conditional Ldlrad3-/- mice. Though the molecular mechanisms of VEEV attachment and entry into host cells during infection are still poorly understood, recently the interaction residues on VEEV have been identified for LDLRAD3 by cryo-electron microscopy. My second Aim will determine which of these contact sites are of functional importance for VEEV infection in vitro and in vivo. Using structure-guided mutagenesis, I will generate VEEV strains encoding mutations at LDLRAD3 interaction sites. These will be tested for altered infection using cell-based assays and differences in pathogenesis using a lethal murine VEEV model. Mutations at LDLRAD3 interaction sites which result in increased VEEV infectivity or binding will be engineered into a LDLRAD3-Fc decoy receptor to optimize its anti- VEEV therapeutic function. Overall, these studies will define how LDLRAD3-dependent tropism of VEEV is critical for the development of clinical disease and what key viral interaction residues required for binding of VEEV to LDLRAD3 might be harnessed for the development of targeted antiviral therapies.

项目概要 甲病毒是披膜病毒科的有包膜正链 RNA 病毒，其自然流行 蚊子媒介和非人类哺乳动物或鸟类物种之间发生循环。委内瑞拉马 脑炎病毒（VEEV）是一种脑炎甲病毒，导致马和人类周期性流行。 美洲各地的人口。它作为雾化生物恐怖主义武器的潜力和 它在研究环境中所造成的风险凸显了采取对策的必要性。 VEEV的发病机制是 其特征是中枢神经系统（CNS）受累和淋巴组织受损。我们的实验室 发表了 VEEV 新型受体的发现，低密度脂蛋白受体 A 类结构域包含 3 (LDLRAD3)，是体内感染和 VEEV 发病机制所必需的。我们已经证明可溶性融合 LDLRAD3 与 IgG Fc 结构域相连的蛋白质 (LDLRAD3-Fc) 可保护小鼠免受致命感染。尽管 这表明，识别进入受体可以成为针对 VEEV 的对策目标。 LDLRAD3 在控制 VEEV 发病机制中的作用仍不清楚。我提案的第一个目标是 探索 LDLRAD3 介导的 VEEV 向性和神经侵袭。我假设 LDLRAD3 是必需的 VEEV 感染周围器官的骨髓细胞和中枢神经系统的神经元，这有助于 炎症和临床疾病的发展。该假设将通过体内感染进行检验 分析感染过程中病毒 RNA 水平、组织病理学和病毒向性的研究 Ldlrad3-/- 小鼠。将使用混合原代探索 LDLRAD3 介导的 VEEV 感染的细胞类型特异性 来自 Ldlrad3-/- 小鼠和组织特异性条件 Ldlrad3-/- 小鼠的神经元培养物。虽然分子 最近，人们对感染期间 VEEV 附着和进入宿主细胞的机制仍知之甚少。 VEEV 上的相互作用残基已通过冷冻电子显微镜鉴定为 LDLRAD3。我的第二个 目标将确定这些接触位点中哪些对于体外和体内 VEEV 感染具有功能重要性。 体内。使用结构引导诱变，我将生成编码 LDLRAD3 突变的 VEEV 菌株 互动网站。这些将使用基于细胞的检测和差异来测试感染的改变 使用致死性小鼠 VEEV 模型研究发病机制。 LDLRAD3 相互作用位点的突变导致 增加的 VEEV 感染性或结合将被设计到 LDLRAD3-Fc 诱饵受体中，以优化其抗 VEEV治疗功能。总体而言，这些研究将定义 VEEV 的 LDLRAD3 依赖性趋向性如何 对于临床疾病的发展至关重要，以及结合所需的关键病毒相互作用残基 VEEV 至 LDLRAD3 可用于开发靶向抗病毒疗法。