Respiratory syncytial virus targets rafts for penetration and immunomodulation

呼吸道合胞病毒以筏为目标进行渗透和免疫调节

• 批准号: 7896496
• 负责人: Homero Gabriel San juan Vergara
• 金额: $ 7.6万
• 依托单位: UNIVERSITY DEL NORTE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-15 至 2013-03-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7896496
• 关键词: Address; Antigen Presentation; Binding; Calcium; Cell Culture Techniques; Cell membrane; Cells; Centrifugation; Cholesterol; Coculture Techniques; Colombia; Confocal Microscopy; Data; Dendritic Cells; Dependence; Detergents; Development; Dominant-Negative Mutation; Dynamin 2; EMSA; Electron Microscopy; Electrophoretic Mobility Shift Assay; Epithelial; Epithelial Cells; Event; Exclusion; Exposure to; Flow Cytometry; Fura-2; GTP-Binding Proteins; Goals; Human; Immune response; Immunofluorescence Immunologic; Infant; Infection; Lovastatin; Lower respiratory tract structure; Measures; Membrane; Membrane Microdomains; Molecular; Morphology; Natural Immunity; Pathway interactions; Penetration; Peptides; Phospho-Specific Antibodies; Phospholipase C; Phosphorylation; Plasmids; Play; Process; Protein Kinase C Alpha; Proteins; Research; Research Personnel; Respiratory Syncytial Virus Infections; Respiratory syncytial virus; Respiratory syncytial virus RSV proteins; Role; Side; Signal Pathway; Signal Transduction; Signaling Molecule; Small Interfering RNA; Source; Sucrose; T-Lymphocyte; Techniques; Testing; Time; Vaccine Therapy; Viral; Virion; Virus; Virus Diseases; Western Blotting; adaptive immunity; base; env Gene Products; immunological synapse; immunoregulation; inhibitor/antagonist; particle; pathogen; prevent; programs; release of sequestered calcium ion into cytoplasm; research study; respiratory; trafficking; virus envelope

DESCRIPTION (provided by applicant): The longer term goal of the research is aimed at finding new ways to prevent or to treat the infection caused by respiratory syncytial virus (RSV). In order to reach such goal, it is required to study the molecular aspects by which the virus enter to the cell, and how the RSV could take advantage of this to modulate the immune response. RSV has been proposed to fuse its envelope directly with the cell membrane; however, cell membrane is not a homogeneous place and that it is constituted by different microdomains such as lipid rafts. The current proposal would help clarify the role that lipid rafts are playing during the viral penetration and the mechanistic aspects involved for allowing the lipid raft to participate. Since lipid rafts cluster very important signaling molecules involved in both innate and adaptive immunity, this proposal would also test the hypothesis that RSV proteins target lipid rafts to modulate the immune response. The specific aims of this project are: 1) To determine whether lipid raft trafficking constitutes a very important pathway for RSV to infect normal human bronchial epithelial (NHBE) cells. To address this aim, lipid rafts will be disrupted by taking away the free membrane cholesterol and RSV infection will be examined in such context. The RSV envelope protein targeting the lipid raft will be examined by evaluating whether RSV-deficient/delta for G and/or SH use lipid rafts to infect NHBE cells. 2) Determine which signaling pathway is involved in assembly of lipid rafts triggered by RSV infection. The role of PKC-alpha, calcium, PI-PLC, and dynamin-2 in the lipid raft clustering triggered by RSV will be examined using pharmacological inhibitors, and dominant negative constructs. 3) Determine whether RSV targets lipid rafts in order to modulate the immune response. NHBE cells transfected with plasmids encoding both EGFP and secreted G (sG) protein will be co-cultured with non-transfected NHBE cells. ERK-1/2 and STAT-1 signaling will be examined using EMSA and anti-phospho-specific antibodies for western-blots and immunocytofluorescence. The effect of the potential presence of RSV proteins in the immunological synapse on the dendritic cell side will be examined by immunofluorescence and confocal microscopy. In Colombia, RSV is the most important respiratory pathogen agent causing 41.8% of the new cases of lower tract respiratory illness in infants. Understanding the mechanistic process of the RSV infection will help in the development of the vaccines and therapies.

描述（由申请人提供）：研究的长期目标旨在寻找预防或治疗由呼吸综合病毒（RSV）引起的感染的新方法。为了达到此类目标，需要研究病毒进入细胞的分子方面，以及RSV如何利用它来调节免疫反应。已提出RSV将其包膜直接与细胞膜融合。然而，细胞膜不是均匀的位置，它是由不同的微域（例如脂质筏）组成的。当前的建议将有助于阐明脂质筏在病毒渗透过程中扮演的角色以及允许脂质筏参与的机械方面。由于脂质筏簇非常重要，因此与先天和适应性免疫有关的信号分子非常重要，因此该提案还将检验以下假设：RSV蛋白靶向脂质筏以调节免疫反应。该项目的具体目的是：1）确定脂质筏运输是否构成RSV感染正常人支气管上皮（NHBE）细胞的非常重要的途径。为了解决这一目标，将在这种情况下检查脂质筏将被剥夺自由膜胆固醇和RSV感染。靶向脂质筏的RSV包膜蛋白将通过评估是否缺乏G和/或SH使用脂质筏感染NHBE细胞的RSV缺陷/三角洲。 2）确定由RSV感染触发的脂质筏的组装中涉及哪种信号通路。 PKC-Alpha，钙，PI-PLC和Dynamin-2在由RSV触发的脂质筏簇中的作用将使用药理抑制剂和显性负构建体检查。 3）确定RSV是否靶向脂质筏以调节免疫反应。用编码EGFP和分泌G（SG）蛋白的质粒转染的NHBE细胞将与未转染的NHBE细胞共培养。 ERK-1/2和STAT-1信号传导将使用EMSA和抗磷酸特异性抗体进行西方印迹和免疫细胞荧光。免疫荧光和共聚焦显微镜检查将检查RSV蛋白在免疫突触侧的潜在存在对树突状细胞侧的影响。在哥伦比亚，RSV是最重要的呼吸病原体剂，导致婴儿较低的婴儿呼吸道疾病的41.8％。了解RSV感染的机械过程将有助于疫苗和疗法的开发。