Clearance of Blood-Borne Arboviruses

血源性虫媒病毒的清除

• 批准号: 10316169
• 负责人: Thomas E Morrison
• 金额: $ 46.55万
• 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 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10316169
• 关键词: Alphavirus; Amino Acids; Antibodies; Arbovirus Infections; Arboviruses; Arginine; Binding; Biochemical; Biological Assay; Blood; Blood Circulation; Cells; Chikungunya virus; 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; Orthobunyavirus; Pathogenesis; Pathogenicity; Pathway interactions; Phagocytes; Population; Positioning Attribute; Post-Translational Protein Processing; Proteins; Proteomics; Risk Factors; Rivers; Role; Severity of illness; Sorting - Cell Movement; Spleen; Surface; System; Tissues; Transgenic Mice; Ubiquitin; Ubiquitination; Vertebrates; Viral; Viral Structural Proteins; Viremia; Virion; Virus; Work; Zika Virus; acquired immunity; antiviral immunity; base; cell type; chikungunya; diphtheria toxin receptor; experimental study; human disease; improved; in vivo; inhibitor; innate immune mechanisms; insight; mouse model; mutant; natural antibodies; new therapeutic target; particle; receptor binding; 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.

项目摘要 虫媒病毒引起严重的人类疾病。虫媒病毒感染脊椎动物后的病毒血症水平是一个关键因素， 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， 虫媒病毒感染后决定病毒血症的因素定义不明确。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）、奥农病毒（ONNV）和寨卡病毒， 通过吞噬细胞的循环。在脾切除小鼠中的实验表明， 用于虫媒病毒清除。相反，病毒在肝脏中积累，清除不依赖于天然抗体 和补体因子，提示非调理剂机制。根据这一想法， 循环甲病毒被清道夫受体（SR）的竞争性抑制剂阻断， 非自身配体和修饰的自身配体的调理摄取。值得注意的是，我们发现单个赖氨酸（K）到精氨酸（R） CHIKV和ONNV的E2糖蛋白（E2 K200 R）以及RRV（E2 K251 R）中的突变被消除， 通过吞噬细胞清除循环甲病毒颗粒，并促进病毒快速传播至远端 组织中此外，用多种其他氨基酸取代CHIKV E2 K200也允许清除 逃避，表明关键K残基和宿主因子之间的特定相互作用。K是后- 翻译修饰（PTM）和病毒体中E2的质谱分析显示，CHIKV E2 K200 是泛素化的。这些实验揭示了一种以前未被认识的控制虫媒病毒的途径 脊椎动物中的病毒血症和传播。我们假设病毒糖蛋白中关键Ks的PTM许可了 通过在肝枯否细胞（KCs）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KCs在病毒清除中的作用 和传播，以及抗病毒免疫的发展。最后，我们将评估吞噬细胞的作用， 细胞，特别是KCs，在虫媒病毒的跨属面板的清除。在具体目标2中， 将定义对SR介导的清除敏感的血液传播虫媒病毒谱。我们将使用ELISA 和细胞结合测定以确定结合病毒颗粒的鼠和人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。具体目标 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 基于光谱的蛋白质组学，以确定用泛素或 其他PTM最后，我们将使用反向遗传学系统的集合来定义特定修饰的基因的作用。 从循环中清除虫媒病毒的Ks。这一工作将为虫媒病毒的机理研究提供新的思路 从流通中清除。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 用于治疗虫媒病毒病。