IDENTIFICATION OF HIV PACKAGING SEQUENCES--GENERATION OF PACKAGING CELL LINES

在这个项目中，我们建议识别以下序列 有效包装人类免疫缺陷病毒所需 病毒(HIV)基因组。在肿瘤病毒中，如RSV或MLV顺式作用 包装所需的序列(但可能不充分) 已在RNA前导序列中鉴定出基因组RNA 位于负链tRNA引物结合位点和 Gag基因的开始。艾滋病毒的类似部分 基因组以及其他5‘基因组区域将在 以确定对HIV基因组包装的影响。 这些序列也将被改造成异源 以确定它们是否赋予 包装专用性。因为人们认为肿瘤病毒Gag基因 产物与前导序列相互作用以促进基因组 包装、纯化的Gag蛋白和突变体 将对GAG基因进行分析，以确定特定的RNA蛋白 相互作用发生在HIV基因组RNA包装中。信息 从这些实验中收集到的信息将被用于 构建高效、无细胞病变的艾滋病毒包装线 可以用来将病毒或其他序列输送到 HIV靶细胞。细菌顺式和反式作用因子的鉴定 艾滋病病毒包装将有助于构建主导地位 抑制病毒的负性突变和反义载体 复制。

In this project we propose to identify the sequences which are required for efficient packaging of the human immunodeficiency virus (HIV) genome. In oncoviruses such as RSV or MLV cis-acting sequences necessary (but perhaps not sufficient) for packaging of genomic RNA have been identified in the RNA leader sequence located between the minus-strand tRNA primer binding site and the start of the gag gene. The analogous portion of the HIV genome, as well as other 5' genomic regions will be deleted in order to determine the effect on packaging of the HIV genome. These sequences will also be engineered into heterologous expression vectors in order to determine whether they confer packaging specificity. Since it is thought that oncoviral gag gene products interact with leader sequences to facilitate genomic packaging, purified gag proteins and mutants engineered in the gag gene will be analyzed to determine if specific RNA-protein interactions occur in HIV genomic RNA packaging. Information gathered from these experiments will be utilized in order to construct efficient, noncytopathic HIV packaging lines which could be utilized in order to deliver viral or other sequences into HIV target cells. Identification of cis-and trans-acting factors in HIV packaging will be useful in the construction of dominant negative mutations and antisense vectors for inhibition of viral replication.