Host Cell Proteins and Hiv Pro-virus Establishment

宿主细胞蛋白和 HIV 前病毒的建立

• 批准号: 6799864
• 负责人: Nikunj V Somia
• 金额: $ 22.28万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6799864
• 关键词: HeLa cells; biotechnology; cell population study; gene mutation; genetic screening; genetic transduction; green fluorescent proteins; high throughput technology; human immunodeficiency virus 1; immunity; immunogenetics; molecular cloning; transfection; transfection /expression vector; virus infection mechanism

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.

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（AIDS）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）已经被药物抑制剂靶向，可以控制病毒引起的病理。然而，病毒的高突变率导致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，因此要考虑的新药物靶点是完成病毒生命周期所需的细胞乳清蛋白。我们的长期目标是描述这些oroteins及其在HIV-1生命周期中的作用。该建议旨在开发一种遗传方法来鉴定这些细胞蛋白。为此，我们提出两个具体目标。1.在人类细胞中分离出抗HIV感染的基因突变体。我们将用一种基于HIV-1的载体来多重感染高度诱变的HeLa细胞群体，该载体转导一种毒性基因。该基因的表达导致细胞死亡。我们将分离和扩大HeLa克隆，生存这种选择，并确认抗性表型。我们将确定该阻断对感染具有特异性，并确定在病毒生命周期中实施阻断的点。2.通过互补克隆拯救克隆的抗性表型。我们将使用一种新的高通量半自动化方法来拯救耐药表型。将表达载体中的17，000个独特的全长cDNA以单孔形式排列在384孔板中。因此，每个克隆的身份和位置是已知的。将这些cDNA克隆以单孔形式转染到特定目的1中分离的突变体克隆中，并将这些转染子用编码荧光素酶的HIV-1载体转导，将鉴定并扩增荧光素酶活性显著增加（超过所有17，000个数据点的平均值）的cDNA克隆。我们将证实（i）cDNA克隆拯救抗性表型的能力，（ii）拯救对感染是特异性的，和（iii）它独特地拯救特异性Hela克隆。