INHIBITING TRANSACTIVATION OF HIV-1

人类免疫缺陷病毒(HIV)是引起 获得性免疫缺陷综合征(艾滋病)。尽管有耗尽的 大多数优先感染艾滋病毒的T4淋巴细胞，有些 细胞可以存活，并可以在较长时间内潜伏感染。 HIV的感染依赖于反式激活因子Tat蛋白，该蛋白 增加从长末端转录的病毒基因的表达 通过与存在于中的顺式反应序列(TAR)相互作用来重复(Ltr) Ltr。从延迟中重新激活可以由TAT本身或由 几种异源DNA中的一种产生的“即刻早期”蛋白质 经常伴随艾滋病毒感染的病毒。不同的DNA元素 可能通过这些即刻的早期激活参与反式激活 蛋白质和TAT。截获艾滋病毒反式激活的优势 抑制蛋白代表了一种旨在阻断艾滋病毒的新方法 在基因调控水平上的感染。一种变异生物的能力 抑制野生型(Wt)蛋白反式激活其a的蛋白质 靶向启动子被称为抑制。抑制突变型蛋白 包括腺病毒E1a在内的三个不同的反式激活基因是 现在已知哪一项有力地支持了TAT压制突变体的预测 可以生成。将使用定点突变来突变TAT 编码区。将从Cys富集区进行保守突变 通过高度碱性区域(密码子22-57)。测试，测试 反式激活，突变的tat基因在外源基因的控制下 启动子，将与HIV LTR-CAT报告质粒在 HeLa和Jurkat细胞。不能反式激活的突变TAT质粒将 与WtTat和HIV LtR-CAT共转染。一个 压制TAT突变体将通过其抑制CAT的能力来鉴定 活性以浓度依赖的方式。随机突变也会 使用简并寡核苷酸和生物筛选方法制备 将用于直接检测HeLa中的静噪表型 细胞，并允许含有突变Tat基因的单个质粒被 获救并进行了测序。已知Ad5的E1a蛋白反式激活 人类免疫缺陷病毒LTR。来自Ad3、Ad5和Adl2的ELA突变体 Ad5早期启动子的激活，将进行压制测试 分别由wt E1a和Tat反式激活HIV LTR。ELA来自 在10%的艾滋病患者中发现的Ad35病毒将被克隆并首先进行测试 用来激活HIV的LTR。然后将对AD35 ELA进行测试 通过在一个区域产生突变来抑制HIV LTR 在其他EIA中保守，并产生静噪表型。突变者 抑制HIV LTR反式激活的TAT或ELA将稳定 在人类细胞系中表达并测试其抑制能力 被艾滋病毒感染。从长远来看，理解两者的细节 HIV LTR的反式激活和抑制可能导致基因方法 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.