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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 拟议的研究是为了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共有的机制，以及使用猫/FIV模型开发针对一般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。该提案的具体目的是：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。这些抗体中的四种阻断糖蛋白/受体相互作用并以CD 134非依赖性方式中和病毒感染。将进行这些抗体表位的定位，结合定点诱变和缺失突变体分析，以帮助确定参与CD 134和CXCR 4结合的分子区域。2)开展CD 134依赖性中和单克隆抗体和含靶表位肽的共结晶研究。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。一旦在特定目标1下的研究中对表位进行了定位，这些研究中也将包括阻断SU与CXCR 4结合的其他表位，这些表位可能或可能或可能不引发CD 134依赖性中和; 3）分析缺乏特定Env可变区的一系列缺失构建体的CD 134、CXCR 4和HSPG结合特性。将所有构建体制备为CHO细胞中的免疫粘附素，并分析结合三种受体类型的能力，如通过FACS分析评估的。一旦确定了最小的受体结合结构域，将通过使用定点诱变来进一步完善后一种分析，以绘制对每个受体相互作用至关重要的残基。含有CD 134最小结合结构域的缺失构建体也将用于目标1下的抗体图谱研究和目标2下的晶体试验。我们还将评估包膜缺失对病毒感染性的影响，在单轮感染的情况下，β-半乳糖苷酶表达FIV工程表达每个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。