INHIBITING TRANSACTIVATION OF HIV-1

人类免疫缺陷病毒（HIV）是艾滋病的主要病原体。 获得性免疫缺陷综合症（艾滋病）。 虽然有损耗， 大多数T4淋巴细胞优先被HIV感染， 细胞存活并可长期保持潜伏感染。 HIV的感染依赖于反式激活因子达特蛋白， 增加从长末端转录的病毒基因的表达 重复序列（LTR）与顺式应答序列（TAR）相互作用， 的LTR。 潜伏期的再激活可能是由达特本身或 由几种异源DNA之一产生的“立即早期”蛋白质 这些病毒经常伴随着HIV感染。 不同的DNA元素 的HIV LTR可能参与这些即时早期转录激活， 蛋白质和达特。一种显性基因对HIV反式激活的阻断 抑制蛋白代表了一种新的方法，旨在阻止艾滋病毒 在基因调控水平上的感染。 一个变种人 蛋白质以抑制野生型（wt）蛋白质反式激活其a 靶向启动子被称为静噪。 静噪突变蛋白 包括腺病毒E1 A在内的三种不同的反式激活基因， 这有力地支持了达特抑制突变体 可以生成。 将使用定点突变来突变达特 编码区将从富含Cys的区域进行保守突变 通过高碱性区域（密码子22 - 57）。以测试 反式激活，突变的达特基因的控制下，一个外来的 启动子，将与HIV LTR-CAT报告基因质粒共转染， HeLa和Jurkat细胞。不能反式激活的突变达特质粒将 用wt达特质粒和HIV LTR-CAT质粒共转染。一 将通过抑制CAT能力来鉴定静噪达特突变体 以浓度依赖的方式。 随机突变也会 使用简并寡核苷酸和生物筛选方法制备 直接测定HeLa细胞的静噪表型 细胞，并允许含有突变达特基因的单个质粒被 拯救并测序 已知Ad 5的E1 A蛋白反式激活 HIV LTR。 来自Ad 3、Ad 5和Ad 12的ElA突变体，其抑制 将测试Ad 5早期启动子的反式激活的抑制 分别通过wt E1 A和达特反式激活HIV LTR。 Ela从 在10%的艾滋病患者中发现的Ad 35病毒将被克隆并首次进行测试 用于HIV LTR的反式激活。 Ad 35 ElA将被检测 通过在一个区域产生突变来抑制HIV LTR， 在其他E1 A中保守，并产生静噪表型。 突变体 抑制HIV LTR反式激活的达特或ElA的表达将稳定地 在人类细胞系中表达，并测试其抑制 艾滋病毒感染。 从长远来看，了解两者的细节 HIV LTR的反式激活和抑制可能导致基因检测方法 治疗和帮助设计治疗剂。

Human immunodeficiency virus (HIV) is the primary etiological agent of acquired immunodeficiency syndrome (AIDS). Although there is depletion of most of the T4 lymphocytes which are preferentially infected by HIV, some cells survive and can remain latently infected for protracted periods. Infection of HIV is dependent upon the transactivator tat protein which increases expression of viral genes transcribed from the long terminal repeat (LTR) by interacting with cis-responsive sequences (TAR) present in the LTR. Reactivation from latency may be mediated by tat itself or by the "immediate early" proteins produced by one of several heterologous DNA viruses which frequently accompanies HIV infection. Different DNA elements of the HIV LTR may be involved in transactivation by these immediate early proteins and tat. Interception of HIV transactivation by a dominant inhibitory protein represents a novel approach aimed at blocking HIV infection at the level of gene regulation. The ability of a mutated protein to inhibit the wild type (wt) protein from transactivating its a target promoter is referred to as squelching. Squelch mutant proteins of three diverse transactivating genes, including E1A of adenovirus (Ad), are now known which strongly supports the prediction that tat squelch mutants can be generated. Site-directed mutagenesis will be used to mutate the tat coding region. Conservative mutations will be made from the Cys-rich region through the highly basic region (codons 22 - 57). To test for transactivation, the mutant tat gene under the control of a foreign promoter, will be cotransfected with an HIV LTR-CAT reporter plasmid in HeLa and Jurkat cells. Mutant tat plasmids which fail to transactivate will be cotransfected with the wt tat plasmid and the HIV LTR-CAT plasmid. A squelch tat mutant will be identified by its ability to inhibit CAT activity in a concentration dependent manner. Random mutations will also be made using degenerate oligonucleotides and a biological screening method will be employed which directly assays for the squelch phenotype in HeLa cells and allows an individual plasmid containing the mutant tat gene to be rescued and sequenced. The E1A protein of Ad5 is known to transactivate the HIV LTR. ElA mutants from Ad3, Ad5 and Adl2 which squelch transactivation of Ad5 early promoters, will be tested for squelching transactivation of the HIV LTR by wt E1A and tat, respectively. ElA from Ad35, virus found in 10% of AIDS patients, will be cloned and first tested for transactivation of the HIV LTR. Ad35 ElA will then be tested for squelching the HIV LTR by generating a mutation in a region that is conserved in other E1As and which produces the squelch phenotype. Mutants of tat or ElA that squelch transactivation of the HIV LTR will be stably expressed in human cell lines and tested for their ability to inhibit infection by HIV. In the long term, understanding the details of both transactivation and squelching of the HIV LTR may lead to methods of gene therapy and help in the design of therapeutic agents.