Host Cell Proteins and Hiv Pro-virus Establishment

描述（由申请人提供）：人类免疫缺陷病毒（HIV）是一种RNA病毒，是导致获得性免疫缺陷综合征（艾滋病）的原因。据估计，这一毁灭性疾病迄今已造成2 000万人死亡，全世界约有3 600万人艾滋病毒呈阳性。在13种病毒编码的蛋白质中，有两种（逆转录酶和蛋白酶）已经成为药物抑制剂的目标，这些药物抑制剂可以控制由病毒引起的病理。然而，病毒的高突变率导致了对这些药物的耐药性。由于大多数病毒是专性细胞内寄生虫，新的药物靶点需要考虑完成病毒生命周期所需的细胞蛋白。我们的长期目标是表征这些蛋白及其在HIV-1生命周期中的作用。这项提议旨在发展一种遗传方法来识别这些细胞蛋白。为此，我们提出两个具体目标。1. 从人类细胞中分离出抗HIV感染的基因突变体。我们将用一种以HIV-1为基础的转导有毒基因的载体，复制感染高度诱变的HeLa细胞群。这种基因的表达会导致细胞死亡。我们将分离和扩增在这种选择中存活的HeLa克隆，并确认抗性表型。我们将确定阻断对感染是特异性的，并确定在病毒生命周期中施加阻断的点。2. 通过互补克隆挽救抗性表型。我们将使用一种新的高通量半自动方法来挽救抗性表型。在384个孔板中以单孔格式排列表达载体中的17000个唯一的全长cdna。因此，每个克隆体的身份和位置都是已知的。这些cDNA克隆将以单孔格式转染到特定目标1中分离的突变克隆中，这些转染物将用编码荧光素酶的HIV-1载体进行转导，荧光素酶活性显着增加（超过所有17,000个数据点的平均值）的cDNA克隆将被识别和扩展。我们将确认(i) cDNA克隆拯救耐药表型的能力，（ii）拯救对感染是特异性的，（iii）它唯一地拯救了特定的Hela克隆。

DESCRIPTION (provided by applicant): Human immunodeficiency virus (HIV) is an RNA virus that is the cause of acquired immunodeficiency syndrome (AIDS). This devastating disease is estimated to have killed 20 million people to date, and approximately 36 million people worldwide are HIV positive. Of the thirteen viral encoded proteins, two (reverse transcriptase and protease) have been targeted by drug inhibitors that can keep the pathology caused by the virus in check. However, the high mutation rate of the virus results in resistance to these drugs. Since most viruses are obligate intracellular parasites, new drug targets to consider are cellular oroteins that are required for completion of the virus life cycle. Our long-term goal is to characterize these oroteins and their role in the HIV-1 life cycle. This proposal is aimed at developing a genetic approach to dentify these cellular proteins. To this end we propose two specific aims. 1. Isolate genetic mutants in human cells that are resistant to infection by HIV. We will multiply infect a highly mutagenized population of HeLa cells with an HIV-1 based vector that transduces a toxic gene. Expression of this gene leads to cell death. We will isolate and expand HeLa clones that survive this selection, and confirm the resistance phenotype. We will establish that the block is specific for infection, and identify the point in the viral life cycle where the block is imposed. 2. Rescue the resistant phenotype of the clones by complementation cloning. We will use a novel high throughput semi-automated approach to rescue the resistance phenotype. 17,000 unique full length cDNAs in expression vectors are arrayed in single well formats in 384 well plates. Hence the identity and position of each clone is known. These cDNA clones will be transfected into the mutant clones isolated in specific aim 1 in single well format and these transfectants will be transduced with an HIV-1 vector that codes for luciferase, cDNA clones that show a significant increase (over the average of all 17,000 datapoints) in luciferase activity will be identified and expanded. We will confirm (i) the ability of the cDNA clone to rescue the resistant phenotype, (ii) that rescue is specific for infection and (iii) that it uniquely rescues specific Hela clones.