Chikungunya Virus Replication and Pathogenesis

 描述(申请人提供)：基孔肯雅病毒(CHIKV)是一种蚊子传播的甲型病毒，会导致一种使人衰弱的肌肉骨骼炎症性疾病的大规模流行。目前还没有批准的针对CHIKV的疫苗或抗病毒药物。CHIKV在E2糖蛋白与宿主细胞表面的糖胺多聚糖(GAG)结合后开始感染，并通过依赖于笼蛋白的摄取促进内化途径。CHIKV减毒株和强毒株都与Gag结合，Gag结合效率影响毒力，包括在CHIKV疾病的小鼠模型中。然而，CHIKV-GAG相互作用的分子基础尚不清楚，这使得人们无法完全了解GAG在CHIKV致病中的作用。此外，促进CHIKV复制的宿主因子、宿主CHIKV感染的确切细胞靶点以及宿主CHIKV复制因子与疾病发病机制之间的联系仍不清楚。这项拟议的研究结合了特伦斯·德莫迪、迈克尔·戴蒙德和托马斯·莫里森的实验室在病毒-受体相互作用、RNA病毒复制、病毒免疫学和病毒疾病小鼠模型方面的专业知识，以增强对CHIKV复制和发病机制的了解。提出了三个综合性、互动性的具体目标。在具体目标1中，将确定CHIKV与GAG结合的机制和病理学意义。CHIKV结合的特定Gag亚型将使用糖链阵列筛选和具有Gag生物合成缺陷的遗传改变的细胞系来定义，与Gag结合所需的E2序列将使用结构引导突变来定义。利用Gag结合能力降低或丧失的CHIKV突变株和Gag基因敲除小鼠，将确定Gag结合在急性和慢性CHIKV疾病中的功能。在特定目标2中，将阐明最近在小干扰RNA筛查中发现的COPI辅酶亚基和调节因子在CHIKV感染中的功能。COPI转运活性减弱的细胞将被CHIKV感染，并测试病毒复制区段的形成、病毒RNA的合成以及病毒后代的组装和释放。将使用新建立的基因靶向小鼠来确定COPI辅助体ARCN1和调节因子GBF1在CHIKV致病中的功能。在具体目标3中，将确定哺乳动物宿主中与CHIKV致病和持久性有关的细胞类型。含有组织特异性microRNA种子序列的CHIKV毒株将在小鼠身上测试急性和慢性疾病，以及趋化因子和细胞因子的阐述，肌肉骨骼组织与炎性白细胞的渗透，以及体液免疫反应的发展。总体而言，这项合作计划的研究将加强我们对CHIKV与GaG结合机制的理解，确定COPI转运在CHIKV感染和发病中的功能，并确定CHIKV靶向宿主中产生疾病的特定细胞。从拟议的研究中获得的知识可能会为抗CHIKV治疗提供新的靶点。

 DESCRIPTION (provided by applicant): Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes massive epidemics of a debilitating musculoskeletal inflammatory disease. There are currently no approved CHIKV-specific vaccines or antiviral agents. CHIKV initiates infection after the E2 glycoprotein binds to glycosaminoglycans (GAGs) on the surface of host cells and promotes internalization by clathrin-dependent uptake into the endocytic pathway. Both attenuated and virulent CHIKV strains bind GAGs, and GAG-binding efficiency influences virulence, including in a mouse model of CHIKV disease. However, the molecular basis of CHIKV-GAG interactions is unknown, which precludes a complete understanding of the function of GAGs in CHIKV pathogenesis. Moreover, the host factors that promote CHIKV replication, the precise cellular targets for CHIKV infection in the host, and links between host CHIKV replication factors and disease pathogenesis also remain unclear. The proposed research combines the expertise of the laboratories of Terence Dermody, Michael Diamond, and Thomas Morrison in virus-receptor interactions, RNA virus replication, viral immunology, and mouse models of viral disease to enhance knowledge of CHIKV replication and pathogenesis. Three integrated and interactive specific aims are proposed. In Specific Aim 1, the mechanisms and pathological significance of CHIKV binding to GAGs will be determined. Specific GAG subtypes bound by CHIKV will be defined using glycan-array screening and genetically altered cell lines with defects in GAG biosynthesis, and sequences in E2 required for binding to GAGs will be defined using structure-guided mutagenesis. The function of GAG binding in acute and chronic CHIKV disease will be determined using mutant CHIKV strains with reduced or abolished GAG-binding capacity and GAG-knockout mice. In Specific Aim 2, the function in CHIKV infection of COPI coatomer subunits and regulatory factors, which were recently identified in a small-interfering RNA screen, will be elucidated. Cells with diminished COPI transport activity will be infected with CHIKV and tested for formation of viral replication compartments, synthesis of viral RNA, and assembly and release of viral progeny. The function of COPI coatomer ARCN1 and regulatory factor GBF1 in CHIKV pathogenesis will be determined using newly established gene-targeted mice. In Specific Aim 3, cell types in the mammalian host that contribute to CHIKV pathogenesis and persistence will be identified. CHIKV strains engineered to contain tissue-specific microRNA seed sequences will be tested for acute and chronic disease in mice and elaboration of chemokines and cytokines, infiltration of musculoskeletal tissues with inflammatory leukocytes, and development of humoral immune responses. Overall, studies in this collaborative proposal will enhance our understanding of mechanisms by which CHIKV binds to GAGs, determine the function of COPI transport in CHIKV infection and pathogenesis, and define specific cells in the host targeted by CHIKV to produce disease. Knowledge gained from the proposed research may illuminate new targets for anti-CHIKV therapeutics.