Clearance of Blood-Borne Arboviruses

血源性虫媒病毒的清除

• 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
• 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' 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

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 虫媒病毒感染脊椎动物后引起严重的人类疾病。 令人惊讶的是，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。 我们发现多种虫媒病毒感染后导致病毒血症的因素尚不明确。 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 在脾脏切除的小鼠中进行的实验表明，脾脏是可有可无的。 相反，病毒在肝脏中积聚，清除率不依赖于天然抗体。 和补体因子，表明与此想法一致的是，清除。 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 值得注意的是，我们发现单个赖氨酸（K）到精氨酸（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 此外，用多种其他氨基酸替代 CHIKV E2 K200 也可以实现清除。 逃避，表明关键 K 残基和宿主因子之间的特定相互作用是后处理的目标。 病毒粒子中 E2 的翻译修饰 (PTM) 和质谱分析表明，CHIKV E2 K200 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。 我们捕获了病毒糖蛋白中关键 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。 我们还将确定捕获循环虫媒病毒的类型，并确定 KC 在病毒清除中的作用。 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，我们在特定目标 2 中进行了研究。 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。 和细胞结合测定，使用 SR 确定结合病毒颗粒的鼠类和人类 SR。 在敲除小鼠中，我们将确定特定 SR 在清除循环虫媒病毒中的作用。 3，我们将定义 E2 泛素化在循环 CHIKV 和 RRV 清除中的作用，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 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。 这项工作将为虫媒病毒从循环中清除提供新的机制认识。 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。