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: 10536362
• 负责人: Natasha Monir Kafai
• 金额: $ 3.27万
• 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/10536362
• 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; 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; Laboratories; Laboratory Personnel; Life; Link; Low Density Lipoprotein Receptor; Lymphoid Tissue; Mammals; Measures; Mediating; Modeling; Molecular; Mus; Mutagenesis; Mutate; Mutation; Myeloid Cells; Neuraxis; Neuroglia; Neurologic; Neurons; Neurotropism; Organ; Pathogenesis; Pathology; Periodicity; 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; Tumor-infiltrating immune cells; Venezuelan Equine Encephalitis Virus; Viral; Viral Load result; Viral Pathogenesis; Viremia; Virus; Virus Diseases; aerosolized; antiviral drug development; base; cell type; cellular targeting; clinical development; decoy receptor 3; improved; in vivo; mouse model; neuroepithelium; novel; prevent; receptor; scavenger receptor; subcutaneous; transcytosis; 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.

项目摘要 甲病毒是披膜病毒科的有包膜的正链RNA病毒，其天然地方性感染是由甲病毒引起的。 在蚊媒和非人类哺乳动物或鸟类之间发生循环。委内瑞拉马 脑炎病毒（VEEV）是一种引起马和人类周期性流行的脑炎甲病毒 美洲各地的人口。其作为雾化生物恐怖主义武器的潜力， 它在研究环境中造成的已证实的风险突出表明需要采取对策。VEEV发病机制是 其特征在于中枢神经系统（CNS）受累和淋巴组织损伤。本实验室 发表了VEEV的一种新受体，低密度脂蛋白受体A类结构域的发现， 3（LDLRAD 3），体内感染和VEEV发病所需。我们已经证明了可溶性融合 具有连接至IgG Fc结构域的LDLRAD 3的蛋白（LDLRAD 3-Fc）保护小鼠免受致死性感染。虽然 这表明，识别进入受体可以成为对抗VEEV的目标， LDLRAD 3在控制VEEV发病机制中的作用仍不清楚。我的建议的第一个目的是 探讨LDLRAD 3介导VEEV的嗜性和神经侵袭。我假设LDLRAD 3是 VEEV感染外周器官中的骨髓细胞和中枢神经系统中的神经元，这有助于 炎症和临床疾病的发展。将使用体内感染来检验这一假设 研究分析了感染过程中病毒RNA水平、组织病理学和病毒嗜性， Ldlrad 3-/-小鼠。将使用混合的原发性支气管肺泡灌洗液（支气管肺泡灌洗液），探索LDLRAD 3介导的VEEV感染的细胞类型特异性。 来自Ldlrad 3-/-小鼠和组织特异性条件性Ldlrad 3-/-小鼠的神经元培养物。虽然分子 VEEV在感染过程中附着和进入宿主细胞的机制目前仍知之甚少， 已经通过冷冻电子显微镜鉴定了VEEV上的相互作用残基与LDLRAD 3的相互作用。我的第二 目的是确定这些接触部位中的哪些对体外和体内VEEV感染具有重要功能。 vivo.使用结构指导诱变，我将产生编码LDLRAD 3突变的VEEV菌株 互动网站将使用基于细胞的测定法检测这些细胞的感染改变， 使用致死性鼠VEEV模型研究其发病机制。LDLRAD 3相互作用位点的突变导致 增加的VEEV感染性或结合将被工程化到LDLRAD 3-Fc诱饵受体中，以优化其抗- VEEV治疗功能。总体而言，这些研究将确定VEEV的LDLRAD 3依赖性向性是如何 对于临床疾病的发展至关重要，以及结合 VEEV与LDLRAD 3的关系可能被用于靶向抗病毒治疗的开发。