MOLECULAR ANALYSIS OF FIV

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这项拟议的研究是为了继续研究猫免疫缺陷病毒(FIV)的基因组特征，以确定进入目标细胞的机制和病毒生命周期的细节。这些研究的最终目的是了解FIV复制的所有方面，特别是与艾滋病毒共有的机制，目的是利用猫科动物/FIV模型制定广泛的干预战略，一般地预防慢病毒感染。这一授权期的重点是确定包膜/受体相互作用的特征，并确定病毒进入靶细胞的分子机制。该提案的具体目的是1)绘制一组识别FIV包膜糖蛋白不同区域的单抗的结合表位图。我们已经生产了一组识别FIV SU的20株单抗。其中四种抗体阻断糖蛋白/受体的相互作用，并以CD134不依赖的方式中和病毒感染。绘制这些抗体表位的图谱，结合定点突变和缺失突变体的分析，将有助于确定CD134和CXCR4结合所涉及的分子区域。2)开展CD134依赖的中和单抗与含靶表位的多肽的共结晶研究。与伊恩·威尔逊博士的实验室合作，我们将与合成和重组多肽以及一个或多个中和单抗进行共结晶实验，以确定该表位周围的局部结构。一旦在特定目的1下的研究中映射了阻止SU与CXCR4结合并且可能或可能不会引起CD134依赖的中和的其他表位，这些研究也将包括在内；以及3)分析一系列缺失结构的CD134、CXCR4和HSPG结合特性，这些缺失构建体缺乏特定的环境可变区。所有构建物都将以免疫粘附素的形式在CHO细胞中制备，并通过流式细胞仪分析，分析其与三种受体类型的结合能力。一旦确定了最小的受体结合域，后一种分析将通过使用定点突变来绘制对每个受体相互作用至关重要的残基图，从而进一步完善后一种分析。含有CD134最小结合域的删除构建体也将用于AIM 1下的抗体图谱研究和AIM 2下的晶体试验。我们还将在单轮感染的情况下评估包膜删除对病毒感染性的影响，该FIV是通过表达每个环境突变株的表达β半乳糖苷酶的FIV来设计的。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的过程。了解病毒进入的机制将有助于开发干预病毒感染的方法。

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.