Chikungunya Virus Replication and Pathogenesis

基孔肯雅病毒复制和发病机制

• 批准号: 9252845
• 负责人: TERENCE S. DERMODY
• 金额: $ 9.71万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9252845
• 关键词: Acute; Acute Disease; Aedes; Affect; Alphavirus; Americas; Anabolism; Antiviral Agents; Arboviruses; Arthritis; Attenuated; Binding; Biochemical; Biological Assay; Biology; Caribbean region; Cell Culture Techniques; Cell Line; Cell membrane; Cell surface; Cell-Matrix Junction; Cells; Chikungunya virus; Chronic; Chronic Disease; Chronic Phase of Disease; Clathrin; Coat Protein Complex I; Collaborations; Colorado; Culicidae; Cytosol; Defect; Development; Diagnosis; Diamond; Disease; Endocytosis; Endoplasmic Reticulum; Endosomes; Engineering; Enzymes; Epidemic; Exanthema; Fever; Future; GBF1 gene; Gene Targeting; Glycoproteins; Glycosaminoglycans; Golgi Apparatus; Health; Human; Immune response; Immunology; Individual; Infection; Infiltration; Inflammatory; Inflammatory Response; Injury; Integration Host Factors; Knockout Mice; Knowledge; Laboratories; Leukocytes; Licensing; Link; Mammalian Cell; Mediating; Mediator of activation protein; Membrane; Membrane Fusion; MicroRNAs; Molecular; Mus; Musculoskeletal; Mutagenesis; North America; Pathogenesis; Pathology; Pathway interactions; Polyarthralgias; Polysaccharides; Property; RNA Viruses; Replicon; Research; Role; Seeds; Site; Small Interfering RNA; Structure; Surface; Testing; Therapeutic; Tissues; Tropism; Universities; Vaccines; Vesicle Transport Pathway; Viral; Viral Pathogenesis; Virion; Virulence; Virulent; Virus; Virus Diseases; Virus Receptors; Virus Replication; Washington; Work; base; cell type; cellular targeting; chemokine; cytokine; genomic RNA; improved; insight; mouse model; mutant; novel therapeutics; public health relevance; research study; screening; therapeutic vaccine; uptake; viral RNA; virus infection mechanism; virus pathogenesis

 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.

 描述（由申请方提供）：基孔肯雅病毒（CHIKV）是一种蚊传播的甲病毒，可导致大规模流行的衰弱性肌肉骨骼炎性疾病。目前没有批准的CHIKV特异性疫苗或抗病毒剂。CHIKV在E2糖蛋白与宿主细胞表面上的糖胺聚糖（GAG）结合后引发感染，并通过网格蛋白依赖性摄取促进内化进入内吞途径。减毒和毒性CHIKV毒株都结合GAG，并且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是未知的，这妨碍了对GAG在CHIKV发病机制中的功能的完全理解。此外，促进CHIKV复制的宿主因子、宿主中CHIKV感染的精确细胞靶标以及宿主CHIKV复制因子与疾病发病机制之间的联系也仍不清楚。拟议的研究结合了Terence Dermody，Michael Diamond和托马斯莫里森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。提出了三个综合和互动的具体目标。在具体目标1中，将确定CHIKV与GAG结合的机制和病理学意义。将使用聚糖阵列筛选和具有GAG生物合成缺陷的遗传改变的细胞系来定义CHIKV结合的特定GAG亚型，并且将使用结构指导的诱变来定义结合GAG所需的E2中的序列。将使用具有降低或消除的GAG结合能力的突变CHIKV株和GAG敲除小鼠来确定GAG结合在急性和慢性CHIKV疾病中的功能。在具体目标2中，将阐明最近在小干扰RNA筛选中鉴定的COPI外壳体亚基和调节因子在CHIKV感染中的功能。将具有降低的COPI转运活性的细胞用CHIKV感染并测试病毒复制区室的形成、病毒RNA的合成以及病毒子代的组装和释放。将使用新建立的基因靶向小鼠确定COPI外被体ARCN 1和调节因子GBF 1在CHIKV发病机制中的功能。在具体目标3中，将鉴定哺乳动物宿主中有助于CHIKV发病机制和持久性的细胞类型。将针对小鼠中的急性和慢性疾病以及趋化因子和细胞因子的产生、炎性白细胞对肌肉骨骼组织的浸润以及体液免疫应答的发展来测试经工程化以含有组织特异性microRNA种子序列的CHIKV菌株。总的来说，这项合作提案中的研究将增强我们对CHIKV与GAG结合的机制的理解，确定COPI转运在CHIKV感染和发病机制中的功能，并确定CHIKV靶向宿主中产生疾病的特定细胞。从拟议研究中获得的知识可能会阐明抗CHIKV治疗的新靶点。