Enveloped Virus Glycoprotein/receptor Interactions

包膜病毒糖蛋白/受体相互作用

• 批准号: 7592209
• 负责人: Edward Berger
• 金额: $ 94.74万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592209
• 关键词: Affect; Antibodies; Antiviral Agents; Asialoglycoprotein Receptor; B-Lymphocytes; Bacteriophages; Biochemical; Biological Assay; CD81 gene; Cell Line; Cell fusion; Cells; Collaborations; Complementary DNA; Cystine; Disease; Flavivirus; G-Protein-Coupled Receptors; Genes; Genome; Glutamates; Glycoproteins; Goals; HIV; Hepatitis C; Hepatitis C virus; Human; Human Herpesvirus 8; Hybridomas; Immunotoxins; Infection; Length; Libraries; Light; Lymphoid Tissue; Lytic Phase; Maintenance; Measurement; Mediating; Monoclonal Antibodies; Mus; National Institute of Child Health and Human Development; Phage Display; Prevention; Production; Proteins; Public Health; RNA; Range; Reagent; Replicon; Reporter; Reporter Genes; Role; Satellite Viruses; Source; Spleen; Structural Genes; Structure; Surface; System; T7 RNA polymerase; Tropism; Vaccines; Variant; Viral; Virion; Virus; Virus-like particle; Washington; Work; base; cell type; design; desire; interest; microbicide; neutralizing antibody; nonhuman primate; novel; receptor; sarcoma; success; tumor

We are analyzing the interactions between virus glycoproteins and their receptors for several enveloped viruses of public health significance in the US and worldwide. Our goals are to define mechanisms of virus entry and cell tropism, identify cellular receptors that mediate virus entry, and develop novel antiviral approaches for treatment (antiviral drugs) and prevention (vaccines, microbicides). 1) Kapsosi's Sarcoma-Associated Virus (KSHV, aka human herpesvirus 8). Our previous work demonstrated that a broad range of human and non-human primate cell types can serve as efficient targets in assays of KSHV glycoprotein-mediated cell fusion and KSHV virion entry. We identified a novel KSHV receptor: xCT, the 12 transmembrane light chain of the xc- cystine/glutamate exchange transport system. During the past year, we focused on obtaining reagents for structure-function studies of the KSHV interaction. To this end, we obtained monoclonal antibodies against xCT from Dr. D. Afar, Protein Design Labs. Two were found to inhibit KSHV cell fusion; curiously, these Mabs did not affect KSHV infection. We have also prepared mg quantities of soluble constructs of KSHV glycoproteins involved in fusion (gH and gB); these will be valuable probes to study gp-receptor interactions. We have also begun collaborative studies with Dr. Leonid Margolis, NICHD, to study KSHV infection in lymphoid tissue explants, with the goals of defining the role of xCT and determining whether this receptor is used for infection of B cells. In a new collaborative effort with Dr. Corey Casper and Dr. Larry Corey, FHCRC, U. Washington, we plan to develop immunotoxins against KSHV proteins for treatment of diseases associated with KSHV infection. For Multicentric Castlemans Disease in which most cells are in the lytic phase, we will make immunotoxins targeting glycoproteins gH, gB, and K8.1A, using previously described or newly generated hybridomas. Approaches will include murine systems (spleen cell fusions, cDNA phage libraries) as well as infected human sources (cDNA phage libraries). For Kaposis sarcoma, we will target the viral G protein coupled receptor which has been implicated in initiation and maintenance of KS tumors. We have initiated a collaboration with Dr. Silvio Gutkind, NIDCR, to produce variant constructs of the vGPCR that yield high surface expression levels; this will facilitate elicitation and analysis of monoclonal antibodies to be used for immunotoxin production. Dr. Ira Pastan, a collaborator in our HIV immunotoxin work, has offered assistance with this effort (though not a formal collaborator). Dr. Robert Purcell has also expressed interest in phage display systems for isolating the desired antibodies. 2) Hepatitis C virus (HCV). We are particularly interested in developing systems to study HCV entry, for use in defining essential receptors and elucidating entry mechanisms, assaying HCV neutralizing antibodies in infected people and in vaccine studies. To this end, we are devising systems to produce HCV virus-like particles containing the functional HCV E1 and E2 glycoproteins and containing an RNA that encodes a reporter gene; this will enable rapid, quantitative, and sensitive measurement of entry. During the past year, we have had considerable success with a novel system in which a full length HCV genome encoding the structural genes but not the nonstructural genes, is introduced into a particular cell line (containing a flavivirus replicon) that produces and secretes infectious (single cycle) HCV virus-like particles. The genome also encodes a component of a novel reporter system based on expression of T7 RNA polymerase from a single RNA molecule introduced by an HCV VLP. We have obtained convincing evidence for VLP entry mediated by the viral E1?E2 glycoproteins, and dependent on previously defined HCV receptors (CD81, asialoglycoprotein receptor, SRBP).

我们正在分析病毒糖蛋白及其受体之间的相互作用，以在美国和世界各地具有公共卫生意义的几种包围病毒。我们的目标是定义病毒进入和细胞卓越的机制，鉴定介导病毒进入的细胞受体，并开发出新型的治疗方法（抗病毒药物）和预防方法（疫苗，微生物剂）。 1）Kapsosi的肉瘤相关病毒（KSHV，又名人类疱疹病毒8）。我们以前的工作表明，在KSHV糖蛋白介导的细胞融合和KSHV病毒体进入的测定中，广泛的人类和非人类灵长类动物细胞类型可以作为有效靶标。我们确定了一种新型的KSHV受体：XCT，XC-胱氨酸/谷氨酸交换传输系统的12个跨膜光链。在过去的一年中，我们专注于获得用于KSHV相互作用的结构功能研究的试剂。为此，我们从D. Afar博士（蛋白质设计实验室）获得了针对XCT的单克隆抗体。发现两个抑制KSHV细胞融合；奇怪的是，这些mAb不会影响KSHV感染。我们还准备了与融合的KSHV糖蛋白的毫克可溶构建体（GH和GB）；这些将是研究GP受体相互作用的宝贵探针。我们还开始与NICHD Leonid Margolis博士进行合作研究，以研究淋巴组织外植体中的KSHV感染，其目标是定义XCT的作用并确定该受体是否用于B细胞的感染。在与美国华盛顿州FHCRC的Corey Casper博士和Larry Corey博士进行的新合作中，我们计划开发针对KSHV蛋白的免疫毒素，以治疗与KSHV感染相关的疾病。对于大多数细胞处于裂解阶段的多中心castlemans病，我们将使用先前描述的或新生成的杂交瘤制造靶向糖蛋白GH，GB和K8.1A的免疫毒素。方法将包括鼠系统（脾细胞融合，cDNA噬菌体库）以及感染的人类来源（cDNA噬菌体文库）。对于Kaposis肉瘤，我们将瞄准病毒G蛋白偶联受体，该受体与KS肿瘤的启动和维持有关。我们已经与NIDCR的Silvio Gutkind博士进行了合作，以生成VGPCR的变异构建体，以产生高表面表达水平。这将促进用于免疫毒素生产的单克隆抗体的诱导和分析。我们的HIV免疫毒素工作的合作者Ira Pastan博士为这项工作提供了帮助（尽管不是正式的合作者）。罗伯特·珀塞尔（Robert Purcell）博士还对间隔离所需抗体的噬菌体显示系统表示了兴趣。 2）丙型肝炎病毒（HCV）。我们特别有兴趣开发用于研究HCV进入的系统，用于定义基本受体和阐明进入机制，并在感染者和疫苗研究中测定HCV中和抗体。为此，我们正在设计系统来生产含有功能性HCV E1和E2糖蛋白的HCV病毒样颗粒，并包含编码报告基因基因的RNA；这将实现进入进入的快速，定量和敏感的测量。在过去的一年中，我们在一个新的系统中取得了巨大的成功，在这个系统中，编码结构基因而不是非结构基因的全长HCV基因组被引入到特定的细胞系（包含Flavivivirus副本）中，并产生并分泌感染性（单个循环）HCV病毒类颗粒。该基因组还基于由HCV VLP引入的单个RNA分子的T7 RNA聚合酶的表达来编码新的报告基因系统的成分。我们已经获得了由病毒E1？E2糖蛋白介导的VLP进入的令人信服的证据，并取决于先前定义的HCV受体（CD81，Asialoglycoprotein受体，SRBP）。