MOLECULAR ANALYSIS OF FIV

FIV 的分子分析

• 批准号: 7957859
• 负责人: John H Elder
• 金额: $ 0.33万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957859
• 关键词: Antibodies; Binding; Biology; Blocking Antibodies; CXCR4 gene; Cell Communication; Cells; Chinese Hamster Ovary Cell; Collaborations; Computer Retrieval of Information on Scientific Projects Database; Coupled; Crystallization; Engineering; Epitopes; Family Felidae; Feline Immunodeficiency Virus; Funding; Fungal Genome; Genome; Glycoproteins; Goals; Grant; HIV; Heparan Sulfate Proteoglycan; Immunoglobulin Variable Region; Infection; Institution; Intervention; Laboratories; Lentivirus Infections; Life Cycle Stages; Maps; Mediating; Molecular; Molecular Analysis; Monoclonal Antibodies; Peptides; Property; Research; Research Personnel; Resources; Series; Site-Directed Mutagenesis; Source; Structure; United States National Institutes of Health; Virus; Virus Diseases; Virus Replication; base; beta-Galactosidase; chemokine receptor; deletion analysis; method development; model development; mutant; neutralizing monoclonal antibodies; receptor; receptor binding; recombinant peptide; research study; virus envelope

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The proposed research is for continuation of studies to characterize the feline immunodeficiency virus (FIV) genome, to define mechanisms of entry into target cells and details of the virus life cycle. The ultimate goal of these studies is to understand all aspects of FIV replication, particularly as regards mechanisms shared with HIV and with the purpose of using the feline/FIV model for development of broad-based intervention strategies against lentivirus infections in general. The focus of this grant period is the characterization of envelop/receptor interactions and defining the molecular mechanisms of virus entry into the target cell. The Specific Aims of the proposal are to 1) Map binding epitopes of a panel of monoclonal antibodies that recognize distinct regions of the FIV envelope glycoprotein. We have produced a panel of 20 monoclonal antibodies that recognize SU of FIV. Four of these antibodies block glycoprotein/receptor interactions and neutralize virus infection in a CD134-independent manner. Mapping of these antibody epitopes, coupled with site-directed mutagenesis and analysis of deletion mutants, will be performed to aid in defining regions of the molecule involved in both CD134 and CXCR4 binding. 2) Carry out Co-crystallization studies of CD134- dependent neutralizing monoclonal antibodies and peptides containing target epitopes. In collaboration with the laboratory of Dr. Ian Wilson, we will perform co-crystallization experiments with synthetic and recombinant peptides and one or more of the neutralizing Mabs to define the local structure around this epitope. Other epitopes that block SU binding to CXCR4 and may or may or may not elicit CD134- dependant neutralization will also be included in these studies, once the epitopes have been mapped in studies under Specific Aim 1; and 3) Analyze the CD134, CXCR4, and HSPG binding properties of a series of deletion constructs lacking specific Env variable regions. All constructs will be prepared as immunoadhesins in CHO cells and analyzed for ability to bind the three receptor types, as assessed by FACS analyses. The latter analyses will be further refined by use of site-directed mutagenesis to map residues critical to each receptor interaction, once minimal receptor binding domains have been identified. Deletion constructs containing minimal binding domains for CD134 will also be utilized in antibody mapping studies under Aim 1 and in crystal trials under Aim 2. We will also assess the influence of envelope deletions on virus infectivity in the context of single round infection by beta-galactosidase-expressing FIV engineered to express each Env mutant. Together, the proposed studies will aid in elucidation of virus/host cell interactions leading to chemokine receptor-mediated entry into the target cell. Understanding the mechanisms of virus entry will aid in development of methods to intervene with virus infection.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 拟议的研究旨在继续研究猫免疫缺陷病毒（FIV）基因组的特征，以确定进入靶细胞的机制和病毒生命周期的细节。这些研究的最终目标是了解 FIV 复制的各个方面，特别是与 HIV 共享的机制，以及使用猫科动物/FIV 模型来制定针对慢病毒感染的广泛干预策略。该资助期的重点是包膜/受体相互作用的表征和定义病毒进入靶细胞的分子机制。该提案的具体目标是 1) 绘制识别 FIV 包膜糖蛋白不同区域的一组单克隆抗体的结合表位。我们已经生产了一组 20 种单克隆抗体，可识别 FIV 的 SU。其中四种抗体可阻断糖蛋白/受体相互作用并以不依赖 CD134 的方式中和病毒感染。这些抗体表位的作图，加上定点诱变和缺失突变体分析，将有助于确定参与 CD134 和 CXCR4 结合的分子区域。 2) 进行CD134依赖性中和单克隆抗体和含有目标表位的肽的共结晶研究。我们将与 Ian Wilson 博士的实验室合作，使用合成和重组肽以及一种或多种中和单克隆抗体进行共结晶实验，以确定该表位周围的局部结构。一旦这些表位已在特定目标 1 下的研究中进行了定位，则阻断 SU 与 CXCR4 结合并可能或可能或可能不会引发 CD134 依赖性中和的其他表位也将包括在这些研究中。 3) 分析一系列缺乏特定 Env 可变区的缺失构建体的 CD134、CXCR4 和 HSPG 结合特性。所有构建体将在 CHO 细胞中制备为免疫粘附素，并通过 FACS 分析评估结合三种受体类型的能力。一旦确定了最小的受体结合域，后面的分析将通过使用定点诱变来绘制对每个受体相互作用至关重要的残基来进一步完善。含有 CD134 最小结合域的删除构建体也将用于目标 1 下的抗体作图研究和目标 2 下的晶体试验。我们还将评估在 β-半乳糖苷酶单轮感染的情况下包膜删除对病毒感染性的影响-表达FIV，经改造以表达每个Env突变体。总之，拟议的研究将有助于阐明病毒/宿主细胞相互作用导致趋化因子受体介导的进入靶细胞。了解病毒进入的机制将有助于开发干预病毒感染的方法。