ENVELOPE GENE PRODUCTS OF MURINE LEUKEMIA VIRUS

这项提议的长期目标是开发逆转录病毒载体。 它可以定向进入特定的细胞类型。逆转录病毒是RNA 病毒是多种癌症和癌症的病原体 免疫缺陷。在病毒的复制过程中，RNA被转化为 双链DNA并整合到宿主染色体中。这 Property使逆转录病毒载体成为引入基因的首选方法 进入细胞。这项建议的重点是了解一种 小鼠白血病(MuLV)进入靶细胞。这是基本的 了解生产性感染的要求是至关重要的。 成功地将病毒靶向特定类型的细胞和 促进使用逆转录病毒载体进行基因治疗。二 在研究中使用了不同宿主范围的逆转录病毒； 亲生性Moloney MulV(M-MuLV)和两性分离物4070A。 逆转录病毒进入是一个复杂的、精心策划的事件。这项建议 剖析了病毒进入的四个步骤。最初的步骤是 病毒膜基因产物(Env)与细胞受体的结合。在……上面 病毒有两种包膜蛋白，表面蛋白(SU)和跨膜蛋白 (TM)蛋白质。病毒宿主范围由N-末端的一半决定 关于苏的。SU的这个区域与两个噬菌体M13涂层融合在一起 蛋白质。逆转录病毒的宿主范围决定因素在 噬菌体表面。嵌合噬菌体的使用允许 受体结合域的快速筛选和诱变。 SU函数的VRB(高变量B)区域稳定 受体特异性复合体。这可以通过稳定一个 SU的构象或通过与受体的二级相互作用。至 解决这些可能性，与VRB接壤的突变是 在大鼠细胞中对温度敏感，但在小鼠细胞中不使用。 生态型受体(阳离子氨基酸)的大鼠同源物 Transporter)已经被分离出来，并且嵌合的小鼠/大鼠受体已经 已生成。与野生型和ts病毒的功能相互作用将是 检查过了。 在受体结合之后，SU内发生构象变化 和TM.这是一个改变蛋白质相互作用的动态过程。穿过 利用第二位点突变，蛋白质与蛋白质的相互作用 将定义TM的胞外结构域。逆转录病毒的最后一步 进入涉及病毒和细胞膜的融合。这个 两性病毒和生态病毒在融合过程中对pH的依赖性不同。 通过使用嵌合的生态型/两性型包膜蛋白， 负责这些不同需求的蛋白质结构域将是 已定义。 该提案的最终目标是制定一个总体方案，以供选择 受体结合域发生变化的包膜蛋白。这导致了 通过新型宿主细胞进入靶向病毒的分离 感受器。

The long term objective of this proposal is to develop retroviral vectors which can target entry into specific cell types. Retroviruses are RNA virus which are the causative agents of a broad range of cancers and immunodeficiencies. In the replication of the virus, the RNA is converted to double-stranded DNA and integrated into the host chromosome. This property has made retroviral vectors a preferred means of introducing genes into cells. The focus of this proposal is to understand athe mechanism of entry of murine Leukemia us (MuLV) into the target cell. This basic understanding of the requirements for a productive infection is essential for the successful targeting of the virus to specific cell types and facilitates the use of retroviral vectors for gene therapy. Two retroviruses with differing host-range are used in the studies; the ecotropic Moloney MulV (M-MuLV) and the amphotropic 4070A isolate. Retroviral entry is a complex, carefully orchestrated event. This proposal dissects four steps in the entry of the virus. The initial step is the binding of the viral envelope gene products (env) to the cell receptor. On the virus are two envelope proteins, the Surface (SU) and the Transmembrane (TM) proteins. The viral host-range is determined by the N-terminal half of SU. This region of SU has been fused with two bacteriophage M13 coat proteins. The host-range determinant of the retrovirus is presented on the surface of the phage. The use of chimeric bacteriophage allows for the rapid screening and mutagenesis of the receptor binding domain. The VRB (highly variable B) region of SU functions to stabilize the receptor specific complex. This can either be through stabilizing a conformation of SU or through secondary interactions with the receptor. To address these possibilities, a mutation bordering the VRB which is temperature-sensitive in Rat cells, but not in mouse cells will be used. The rat homolog of the ecotropic receptor (the cationic amino acid transporter) has been isolated and chimeric mouse/rat receptors have ben generated. Functional interactions with the wild type and ts virus will be examined. Subsequent to receptor binding, conformation changes occur within the SU and TM. This is a dynamic process altering protein interactions. Through the use of second-site mutations, the protein-protein interactions of the extracellular domain of TM will be defined. The final step of retroviral entry involves the fusion of the viral and cellular membranes. The amphotropic and ecotropic virus differ in their pH dependence for fusion. Through the use of chimeric ecotropic/amphotropic envelope proteins, the domain of the proteins responsible for these divergent requirements will be defined. The final aim of this proposal develops a general scheme to select for envelope proteins with altered receptor binding domains. This lead to the isolation of virus which target athe entry through novel host cell receptors.