THE ENVELOPE GENE PRODUCTS OF MURINE LEUKEMIA VIRUS

鼠白血病病毒包膜基因产物

• 批准号: 3194281
• 负责人: MONICA J ROTH
• 金额: $ 13.53万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 1994-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3194281
• 关键词: Retroviridae disease; cysteine; gene therapy; glycosylation; host organism interaction; murine leukemia virus; protein structure function; site directed mutagenesis; virus envelope; virus genetics; virus infection mechanism; virus protein; virus replication

Retroviruses are RNA viruses capable of inducing various diseases including cancers and immunodeficiencies. Many retroviruses cause cellular transformation because they carry oncogenes transduced from cellular genes. Due to their high efficiency in transferring genes, retroviral vectors, ironically, are a preferred means of introducing genes into cells for gene therapy. The long-term goal of this proposal is to understand on the molecular level the mechanism by which Murine Leukemia Viruses (MuLV) choose and enter their host cells. The viral gene products which are known to be required for the entry of the virus into the cells (env gene products) will be studied genetically and biochemically. Identification of the various functional domains of the env gene products provides the basis for future experiments aimed at targeting the entry of the virus to specific cell types and facilitates the use of retroviruses for gene therapy. The env gene products from two MuLV isolates with differing host-range will be analyzed and compared. Linker-insertion mutations will be generated throughout the env gene of the Moloney ecotropic MuLV, which only infects rodent cells, as well as the amphotropic 4070A isolate, which can infect many mammalian species. The effects of the mutations on the viral life-cycle will be determined. Functional domains within the proteins will be localized by the clustering of mutations with identical phenotypes. Regions required for correct processing and transport to the cell surface, association with the viral particle, syncytium formation, and receptor binding may be identified. A second approach to identify regions required for receptor binding and recognition will involve the generation of hybrid amphotropic/ecotropic env gene products. Variations within the env gene alter the host-range of the virus and direct the entry using differing host-cell receptors. Hybrid env molecules in which the majority of the amphotropic env gene has replaced the ecotropic gene has produced virus with the amphotropic host- range. Using cloning techniques, various sections of the amphotropic env gene will be systematically introduced into the ecotropic backbone. The host-range of the hybrid gene product will be examined and regions required for the receptor binding and tropism will be determined. env gene products with altered oligosaccharide moieties will be generated to address the role of the multiple glycosylations. The potential glycosylation sites will be changed using oligonucleotide-directed mutagenesis. The effects of the loss of the individual sites as well as multiple glycosylation sites will be examined. The final processed form of the env gene product contains two proteins, SU and TM, are covalently bound through a disulfide bridge. the cysteine residue involved in this bond in the TM protein will be identified.

逆转录病毒是能够引起各种疾病的rna病毒。 包括癌症和免疫缺陷。许多逆转录病毒导致 细胞转化，因为它们携带从 细胞基因。由于它们在转移基因方面的高效率， 具有讽刺意味的是，逆转录病毒载体是一种引入基因的首选方法 转化成细胞进行基因治疗。这项提议的长期目标是 从分子水平认识小鼠白血病的发病机制 病毒(MuLV)选择并进入其宿主细胞。病毒基因产品 已知是病毒进入细胞所必需的 (env基因产物)将进行遗传和生化研究。 Env基因产物各功能结构域的鉴定 为未来的实验提供了基础，这些实验旨在针对 病毒对特定细胞类型的作用，并便于逆转录病毒的使用 用于基因治疗。 两株寄主范围不同的弱毒株的env基因产物 将被分析和比较。链接器插入突变将是 在Moloney生态型MuLV的整个env基因中产生 只感染啮齿动物细胞以及两性菌株4070A，该菌株 可以感染许多哺乳动物物种。基因突变对人类免疫功能的影响 病毒的生命周期将被确定。中的功能域 蛋白质将通过具有相同突变的聚类来定位 表型。正确处理和传输到 细胞表面，与病毒颗粒，合胞体形成， 并且可以识别受体结合。 确定受体结合所需区域的第二种方法和 认识将涉及产生两性/生态性混合 Env基因产物。Env基因内的变异改变了 病毒并使用不同的宿主细胞受体引导进入。 一种杂交膜分子，其中大多数两性膜基因具有 用两性宿主取代了生态型基因产生了病毒- 射程。利用克隆技术，两性环境的不同部分 基因将被系统地导入到生态型主干中。这个 杂交基因产物的寄主范围将被检查和区域 所需的受体结合和取向将被确定。 将产生寡糖部分改变的env基因产物 以解决多重糖基化的作用。潜力 糖基化位点将通过使用寡核苷酸来改变 诱变。失去个别遗址的影响以及 将检查多个糖基化位点。 Env基因产物的最终加工形式包含两种蛋白质，Su 和TM，通过二硫键共价结合。半胱氨酸 TM蛋白中这个键中的残基将被识别出来。