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Clearance of Blood-Borne Arboviruses

血源性虫媒病毒的清除

基本信息

批准号：
10532194
负责人：
Thomas E Morrison
金额：
$ 12.05万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-21 至 2025-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10532194
关键词：
Alphavirus Amino Acids Antibodies Arbovirus Infections Arboviruses Arginine Binding Biochemical Biological Assay Blood Cells Chikungunya virus Circulation Collection Complement Data Development Disease Distal Enzyme-Linked Immunosorbent Assay Flavivirus Geography Glycoproteins Goals Human Immunoglobulin G Immunoglobulin M Immunohistochemistry In Situ Hybridization Individual Integration Host Factors Knockout Mice Knowledge Kupffer Cells Licensing Ligands Liver Lysine Mass Spectrum Analysis Mediating Mus Mutation Mutation Analysis O&apos nyong-nyong virus Orthobunyavirus Pathogenesis Pathogenicity Pathway interactions Phagocytes Population Positioning Attribute Post-Translational Protein Processing Predisposition Proteins Proteomics Risk Factors Role Severity of illness Sorting Spleen Surface System Tissues Transgenic Mice Ubiquitin Ubiquitination Vertebrates Viral Viral Structural Proteins Viremia Virion Virus Work Zika Virus acquired immunity antiviral drug development antiviral immunity arboviral disease cell type diphtheria toxin receptor experimental study human disease improved in vivo inhibitor innate immune mechanisms insight mouse model mutant natural antibodies new therapeutic target particle reverse genetics scavenger receptor therapy development transmission process uptake viral RNA viral transmission

项目摘要

PROJECT SUMMARY Arboviruses cause serious human disease. Viremia level following arbovirus infection of vertebrates is a critical determinant of viral transmission cycles, global viral spread, and disease severity in individuals. Surprisingly, the factors that dictate viremia following arbovirus infection are poorly defined. We found that multiple arboviruses, including chikungunya (CHIKV), Ross River (RRV), o’nyong nyong (ONNV) and Zika viruses, are cleared from the circulation by phagocytic cells. Experiments in splenectomized mice showed that the spleen is dispensable for arboviral clearance. Instead, virus accumulates in the liver and clearance is independent of natural antibodies and complement factors, suggesting a non-opsonic mechanism. Consistent with this idea, clearance of circulating alphaviruses was blocked by competitive inhibitors of scavenger receptors (SRs) that mediate non- opsonic uptake of non-self and modified-self ligands. Remarkably, we found that single lysine (K) to arginine (R) mutations in the E2 glycoproteins of CHIKV and ONNV (E2 K200R), as well as RRV (E2 K251R), abrogated clearance of circulating alphavirus particles by phagocytic cells, and promoted rapid viral dissemination to distal tissues. Moreover, substitution of CHIKV E2 K200 with a variety of other amino acids also allows for clearance evasion, suggesting a specific interaction between key K residues and a host factor. Ks are targets for post- translational modification (PTM), and mass spectrometry analysis of E2 in virions revealed that CHIKV E2 K200 is ubiquitinated. These experiments have revealed a previously unrecognized pathway that controls arbovirus viremia and dissemination in vertebrates. We hypothesize that PTM of key Ks in viral glycoproteins licenses the capture of circulating arboviruses via SRs expressed on liver Kupffer cells (KCs). In Specific Aim 1, the cell types that capture circulating arboviruses will be defined. We also will determine the role of KCs in viral clearance and dissemination, and the development of anti-viral immunity. Finally, we will evaluate the role of phagocytic cells, and KCs specifically, in the clearance of a genera-spanning panel of arboviruses. In Specific Aim 2, we will define the spectrum of blood-borne arboviruses susceptible to SR-mediated clearance. We will use ELISA and cellular binding assays to determine the murine and human SR(s) that bind virus particles. Using SR knockout mice, we will determine the role of specific SRs in clearance of circulating arboviruses. In Specific Aim 3, we will define the role of E2 ubiquitination in the clearance of circulating CHIKV and RRV. We will use mass spectrometry-based proteomics to determine K residues in arboviral particles that are modified with ubiquitin or other PTMs. Finally, we will use a collection of reverse genetics systems to define the role of specific modified Ks in arboviral clearance from the circulation. This work will provide new mechanistic understanding of arbovirus clearance from the circulation. Elucidating these mechanisms could provide new insight into viral transmission, dissemination, and pathogenesis, identify new risk factors of severe disease, and reveal new therapeutic targets for the treatment of arboviral disease.

项目概要虫媒病毒会引起严重的人类疾病。脊椎动物虫媒病毒感染后的病毒血症水平是一个关键病毒传播周期、全球病毒传播和个体疾病严重程度的决定因素。令人惊讶的是，虫媒病毒感染后导致病毒血症的因素尚不清楚。我们发现多种虫媒病毒，包括基孔肯雅病毒 (CHIKV)、罗斯河病毒 (RRV)、奥尼永尼永病毒 (ONNV) 和寨卡病毒，均已从吞噬细胞的循环。脾脏切除小鼠实验表明，脾脏是可有可无的用于清除虫媒病毒。相反，病毒在肝脏中积聚，清除率独立于天然抗体和补体因子，表明非调理性机制。与这个想法相一致，循环的甲病毒被清道夫受体（SR）的竞争性抑制剂阻断，该抑制剂介导非非自配体和修饰自配体的调理摄取。值得注意的是，我们发现单个赖氨酸 (K) 与精氨酸 (R) CHIKV 和 ONNV (E2 K200R) 以及 RRV (E2 K251R) 的 E2 糖蛋白突变被废除吞噬细胞清除循环的甲病毒颗粒，并促进病毒快速传播到远端组织。此外，用多种其他氨基酸取代 CHIKV E2 K200 也可实现清除逃避，表明关键 K 残基和宿主因子之间存在特定的相互作用。 Ks 是后目标病毒粒子中 E2 的翻译修饰 (PTM) 和质谱分析表明，CHIKV E2 K200 是泛素化的。这些实验揭示了一种以前未被识别的控制虫媒病毒的途径病毒血症和在脊椎动物中的传播。我们假设病毒糖蛋白中关键 K 的 PTM 许可通过肝库普弗细胞 (KC) 上表达的 SR 捕获循环虫媒病毒。在具体目标 1 中，细胞将定义捕获循环虫媒病毒的类型。我们还将确定 KC 在病毒清除中的作用和传播以及抗病毒免疫力的发展。最后，我们将评估吞噬细胞的作用细胞，特别是 KC，在跨属虫媒病毒组的清除中发挥作用。在具体目标 2 中，我们将定义易受 SR 介导清除影响的血源性虫媒病毒谱。我们将使用 ELISA 以及细胞结合测定，以确定结合病毒颗粒的鼠类和人类 SR。使用SR 敲除小鼠，我们将确定特定 SR 在清除循环虫媒病毒中的作用。特定目标 3，我们将定义E2泛素化在清除循环CHIKV和RRV中的作用。我们将使用质量基于光谱分析的蛋白质组学，用于确定用泛素或修饰的虫媒病毒颗粒中的 K 残基其他 PTM。最后，我们将使用一系列反向遗传学系统来定义特定修饰的作用 Ks 清除循环中的虫媒病毒。这项工作将为虫媒病毒提供新的机制理解从循环中清除。阐明这些机制可以为病毒传播提供新的见解，传播和发病机制，确定严重疾病的新危险因素，并揭示新的治疗靶点用于治疗虫媒病毒疾病。