Clearance of Blood-Borne Arboviruses

血源性虫媒病毒的清除

基本信息

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
项目类别：
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

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.

项目摘要 arbovirus会导致严重的人类疾病。脊椎动物arbovirus感染后的病毒血症水平是关键病毒传播周期，全球病毒扩散和疾病严重程度的决定因素。令人惊讶的是， arbovirus感染后决定病毒血症的因素的定义很差。我们发现多个arboviruse，包括Chikungunya（Chikv），Ross River（RRV），O'Nyong Nyong（Onnv）和Zika病毒，从吞噬细胞的循环。脾切除小鼠的实验表明脾脏是可分配的用于arboviral清除。取而代之的是，病毒积聚在肝脏中，清除与天然抗体无关和补体因素，提示非上官机制。与这个想法一致循环α病毒被介导非 - 对非自身和改良自我配体的opsonic摄取。值得注意的是，我们发现单赖氨酸（K）到精氨酸（R） CHIKV和ONNV（E2 K200R）的E2糖蛋白以及RRV（E2 K251R）的突变，废除通过吞噬细胞清除循环α病毒颗粒，并促进了远端的快速病毒传播组织。此外，用各种其他氨基酸替换Chikv E2 K200也可以清除逃避，表明关键K残基与宿主因子之间存在特定的相互作用。 KS是后的目标 E2中E2的转化修饰（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先前无法识别的途径，可以控制Arbovirus 病毒血症和脊椎动物的传播。我们假设病毒糖蛋白中的关键KS的PTM许可通过在肝脏库普弗细胞（KCS）上表达的SR捕获循环的arbovirus。在特定的目标1中，单元格将定义捕获循环弧病毒的类型。我们还将确定KC在病毒清除中的作用和传播，以及抗病毒免疫的发展。最后，我们将评估吞噬细胞的作用细胞和KCS专门在清除跨孢病毒属属面板时。在特定的目标2中，我们将定义易受SR介导的清除率的血传播arbovirus的频谱。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类SR。使用SR 敲除小鼠，我们将确定特定SR在清除循环弧病毒中的作用。在特定目标中 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法的蛋白质组学来确定用泛素或用泛素修饰或其他PTM。最后，我们将使用反向遗传系统的集合来定义特定修改的作用循环中的Arboviral清除中的KS。这项工作将提供对Arbovirus的新机械理解循环清除。阐明这些机制可以为病毒传播提供新的见解，传播和发病机理，识别严重疾病的新危险因素，并揭示了新的治疗靶标用于治疗灰烬病毒疾病。