THE ENVELOPE GENE PRODUCTS OF MURINE LEUKEMIA VIRUS

鼠白血病病毒包膜基因产物

• 批准号: 2093518
• 负责人: MONICA J ROTH
• 金额: $ 14.06万
• 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 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2093518
• 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.

逆转录病毒是一种RNA病毒，能够诱导多种疾病 包括癌症和免疫缺陷。 许多逆转录病毒引起 细胞转化，因为它们携带癌基因， 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，逆转录病毒载体是引入基因的优选手段， 用于基因治疗 该提案的长期目标是 在分子水平上理解小鼠白血病 病毒（MuLV）选择并进入它们的宿主细胞。 病毒基因产物 已知病毒进入细胞所需的蛋白质 (env基因产物）将进行遗传和生物化学研究。 env基因产物不同功能域的鉴定 为今后的实验提供了基础， 病毒的特定细胞类型，并促进逆转录病毒的使用 基因治疗 来自两个不同宿主范围的MuLV分离物的env基因产物 将进行分析和比较。 接头插入突变将是 在整个莫洛尼亲嗜性MuLV的env基因中产生， 只感染啮齿动物细胞，以及嗜中性4070 A分离株， 可以感染很多哺乳动物 基因突变对 将确定病毒的生命周期。 功能域内的 蛋白质将通过具有相同突变的突变聚类来定位， 表型 正确处理和运输到 细胞表面，与病毒颗粒结合，合胞体形成， 并且可以鉴定受体结合。 第二种方法是鉴定受体结合所需的区域， 识别将涉及杂交嗜热/嗜亲的产生 env基因产物。 env基因内的变异改变了 并使用不同的宿主细胞受体引导病毒进入。 杂合env分子，其中大多数亲嗜性env基因具有 取代亲嗜性基因的是亲嗜性宿主产生的病毒 范围 利用克隆技术， 基因将被系统地引入亲嗜性骨架。 的 将检查杂交基因产物的宿主范围， 将确定受体结合所需的量和向性。 将产生具有改变的寡糖部分的env基因产物 来阐明多重糖基化的作用。 的潜在 糖基化位点将被改变， 诱变 失去个别场址的影响以及 将检查多个糖基化位点。 env基因产物的最终加工形式含有两种蛋白质，SU 和TM通过二硫桥共价结合。 的半胱氨酸 将鉴定TM蛋白中参与该键的残基。