Segal David J, Gonçalves João, Eberhardy Scott, Swan Christina H, Torbett Bruce E, Li Xuelin, Barbas Carlos F
The Skaggs Institute for Chemical Biology and the Departments of Molecular Biology and Chemistry, The Scripps Research Institute, La Jolla, California 92037, USA.
J Biol Chem. 2004 Apr 9;279(15):14509-19. doi: 10.1074/jbc.M400349200. Epub 2004 Jan 20.
Small molecule inhibitors of human immunodeficiency virus, type 1 (HIV-1) have been extremely successful but are associated with a myriad of undesirable effects and require lifelong daily dosing. In this study we explore an alternative approach, that of inducing intracellular immunity using designed, zinc finger-based transcription factors. Three transcriptional repression proteins were engineered to bind sites in the HIV-1 promoter that were expected to be both accessible in chromatin structure and highly conserved in sequence structure among the various HIV-1 subgroups. Transient transfection assays identified one factor, KRAB-HLTR3, as being able to achieve 100-fold repression of an HIV-1 promoter. Specificity of repression was demonstrated by the lack of repression of other promoters. This factor was further shown to repress the replication of several HIV-1 viral strains 10- to 100-fold in T-cell lines and primary human peripheral blood mononuclear cells. Repression was observed for at least 18 days with no significant cytotoxicity. Stable T-cell lines expressing the factor also do not show obvious signs of cytotoxicity. These characteristics present KRAB-HLTR3 as an attractive candidate for development in an intracellular immunization strategy for anti-HIV-1 therapy.
1型人类免疫缺陷病毒（HIV-1）的小分子抑制剂已经取得了巨大成功，但也伴随着众多不良影响，并且需要每日终身服药。在本研究中，我们探索了一种替代方法，即使用设计的基于锌指的转录因子诱导细胞内免疫。构建了三种转录抑制蛋白，使其结合HIV-1启动子中的位点，这些位点预计在染色质结构中易于接近，并且在各种HIV-1亚组的序列结构中高度保守。瞬时转染试验确定了一种因子KRAB-HLTR3能够实现对HIV-1启动子100倍的抑制。对其他启动子缺乏抑制作用证明了抑制的特异性。该因子进一步显示在T细胞系和原代人外周血单核细胞中对几种HIV-1病毒株的复制有10到100倍的抑制作用。观察到抑制作用至少持续18天，且无明显细胞毒性。表达该因子的稳定T细胞系也未显示出明显的细胞毒性迹象。这些特性使KRAB-HLTR3成为抗HIV-1治疗细胞内免疫策略开发中一个有吸引力的候选者。
