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.

项目摘要 α病毒是togaviridae家族的阳性链RNA病毒，其自然流行 周期发生在蚊子载体与非人类哺乳动物或禽类之间。委内瑞拉马 脑炎病毒（VEEV）是一种负责马和人类周期性流行病的脑α病毒 整个美洲的人口。它有可能用作雾化的生物恐怖武器和 它在研究环境中构成的风险表明了对对策的需求。 Veev发病机理是 以中枢神经系统（CNS）的参与和对淋巴组织的损害为特征。我们的实验室 发表了一种发现VEEV的新型受体，低密度脂蛋白受体A类包含 3（ldlrad3），体内感染和VEEV发病机理所必需的。我们已经证明了可溶的融合 与IgG FC结构域（LDLRAD3-FC）相关的LDLRAD3蛋白质可保护小鼠免受致命感染的影响。尽管 这表明，进入受体的识别可以成为针对Veev的对策的目标 LDLAD3在控制VEEV发病机理中的作用仍不清楚。我提议的第一个目的将 探索VEEV的LDLRAD3介导的tropism和神经入侵。我假设ldlrad3是必需的 中枢神经系统中周围器官和神经元中髓样细胞的VEEV感染，这有助于 炎症和临床疾病的发展。该假设将使用体内感染进行检验 分析病毒RNA水平，组织病理学和病毒疗法在感染期间感染期间的研究 ldlrad3 - / - 小鼠。 LDLAD3介导的VEEV感染的细胞类型特异性将使用混合主要原发性探索 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，VEEV附着和进入宿主细胞进入宿主细胞的机制仍然很少了解，最近 通过冷冻电子显微镜为LDLRAD3鉴定了VEEV上的相互作用残基。我的第二个 AIM将确定哪些接触位点对VEEV感染的体外和IN具有功能重要性 体内。使用结构引导的诱变，我将生成编码LDLAD3突变的VEEV菌株 相互作用位点。这些将通过基于细胞的测定和差异来测试这些改变感染的感染。 使用致命的鼠VEEV模型的发病机理。 LDLAD3相互作用位点的突变，导致 VEEV感染性或结合的提高将被设计到LDLRAD3-FC诱饵受体中，以优化其抗抗诱导受体 VEEV治疗功能。总体而言，这些研究将定义VEEV的LDLRAD3依赖性的趋势是如何 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LDLAD3可能是为了开发靶向抗病毒疗法而利用的。