Clearance of Blood-Borne Arboviruses

项目总结 虫媒病毒会导致严重的人类疾病。脊椎动物感染虫媒病毒后的病毒血症水平是一个关键 病毒传播周期、全球病毒传播和个人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未明确。我们发现了多种虫媒病毒， 包括基孔肯雅(CHIKV)、罗斯河(RRV)、O‘nyong Nyong(ONNV)和寨卡病毒 吞噬细胞的循环。脾切除小鼠的实验表明，脾是可有可无的 进行树枝病毒清除。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，暗示了一种非调理机制。与这一想法一致的是，清除 循环中的甲型病毒被清道夫受体(SRS)的竞争性抑制剂阻断，SRS介导非 非自体和修饰自体配体的光学摄取。值得注意的是，我们发现单一赖氨酸(K)到精氨酸(R) CHIKV和ONNV(E2 K200R)以及RRV(E2 K251R)的E2糖蛋白突变被取消 吞噬细胞清除循环中的甲型病毒颗粒，并促进病毒向远端的快速传播 纸巾。此外，用多种其他氨基酸替代CHIKV E2 K200也允许清除 逃避，提示关键K残基与宿主因子之间存在特定的相互作用。KS是后处理的目标 翻译修饰(PTM)和对病毒粒子中E2的质谱分析表明CHIKV E2 K200 无处不在。这些实验揭示了一种以前未知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 通过表达在肝脏枯否细胞(KCs)上的SRS捕获循环虫媒病毒。在特定目标1中，细胞 将定义捕获传播的虫媒病毒的类型。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，清除跨越虫媒病毒属的小组。在具体目标2中，我们 将确定易受SR介导的清除的血媒虫媒病毒的谱。我们将使用ELISA卡 细胞结合试验确定与病毒颗粒结合的小鼠和人SR(S)。使用SR 在基因敲除小鼠中，我们将确定特定SRs在清除循环虫媒病毒中的作用。以特定的目标 3，我们将确定E2泛素化在清除循环CHIKV和RRV中的作用。我们将使用质量 基于光谱的蛋白质组学测定泛素或泛素修饰的虫媒病毒颗粒中的K残留量 其他PTM。最后，我们将使用一组反向遗传学系统来定义特定修饰的作用 KS从循环中清除了虫媒病毒。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，并揭示新的治疗目标 用于治疗虫媒病毒病。

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.