ENVELOPE GENE PRODUCTS OF MURINE LEUKEMIA VIRUS

这项提案的长期目标是开发逆转录病毒载体 其可以靶向进入特定细胞类型。 逆转录病毒是RNA 病毒，它们是多种癌症的病原体， 免疫缺陷 在病毒的复制过程中， 双链DNA并整合到宿主染色体中。 这 这种特性使逆转录病毒载体成为导入基因的首选方法 转化成细胞 本建议的重点是了解 小鼠白血病病毒（MuLV）进入靶细胞。 这一基本 了解有效感染的要求至关重要 使病毒成功靶向特定细胞类型， 有助于逆转录病毒载体用于基因治疗。 两 研究中使用了具有不同宿主范围的逆转录病毒; 嗜亲性Moloney MulV（M-MuLV）和嗜亲性4070 A分离物。 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。 第一步是 病毒包膜基因产物（env）与细胞受体的结合。 对 病毒有两种包膜蛋白，表面蛋白（SU）和跨膜蛋白（Transmembrane (TM)proteins. 病毒的宿主范围由N-末端的一半决定 的SU。 SU的这一区域与两个噬菌体M13外壳融合， proteins. 逆转录病毒的宿主范围决定因子见 噬菌体的表面。 嵌合噬菌体的使用允许 受体结合结构域的快速筛选和诱变。 SU的VRB（高度可变B）区域的功能是稳定 受体特异性复合物 这可以通过稳定 SU构象或通过与受体的次级相互作用。 到 解决这些可能性，VRB边界的突变， 将使用在大鼠细胞中对温度敏感，但在小鼠细胞中不敏感的。 亲性受体的大鼠同系物（阳离子氨基酸 转运蛋白）已被分离，嵌合小鼠/大鼠受体已被分离， 生成的. 与野生型和ts病毒的功能性相互作用将是 考察 受体结合后，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。 通过 第二个位点突变的使用，蛋白质-蛋白质相互作用， 将定义TM的细胞外结构域。 逆转录病毒的最后一步 进入包括病毒和细胞膜的融合。 的 嗜热性和嗜亲性病毒在它们对融合的pH依赖性方面不同。 通过使用嵌合的亲嗜性/兼嗜性包膜蛋白， 负责这些不同要求的蛋白质的结构域将是 定义了 本建议的最终目的是制定一个通用的方案， 具有改变的受体结合域的包膜蛋白。 这导致了 靶向通过新宿主细胞进入宿主细胞病毒的分离 受体。

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.