Establishment of HIV-resistant lymphocytes by using retrovirus-vector expressing antisense RNA for Tat.

在本项目中，基于在艾滋病中被HIV杀死的cd4阳性细胞在消灭病毒中起主要作用这一事实，试图建立能够抵抗人类免疫缺陷病毒（HIV）感染的cd4阳性淋巴细胞。首先，我们研究了引入表达反义RNA的质粒对由强金属硫蛋白启动子衍生的Tat和Rev的影响。用表达HIV-LTR-CAT和tat的质粒转染人cd4阳性T细胞株MOLT4。瞬时CAT检测发现，与表达Tat和Rev反义RNA的质粒共转染可显著阻断Tat对HIV-LTR的反式激活。然后，我们选择稳定转染了tat -反义RNA质粒的MOLT4细胞进行进一步的生物学研究。然而，由于这些细胞克隆的生存能力不足以证明对HIV感染的实际抗性，因此尚未获得实质性的证据。除上述实验外，逆转录病毒载体的构建正在进行中，该载体能够赋予细胞产生针对HIV Tat（和Rev）的反义RNA的能力。由于这种逆转录病毒载体也含有新霉素耐药基因，因此选择和建立产生反义RNA的细胞现在变得容易得多。我们正急切地尝试测试这些细胞是否能抵抗HIV感染。

In this project, attempt have been made to establish CD4-positive lymphocytes which are resistant to the infection by human immunodeficiency virus (HIV) based on the fact that CD4-positive cells which are killed by HIV in AIDS play a major role in elimination of viruses.First, we have investigated the effect of introduction of the plasmid expressing antisense RNA against Tat and Rev derived by a strong metallothioneine promoter. A human CD4-positive T cell line, MOLT4, was transfected with HIV-LTR-CAT and Tat-expression plasmids. Trans-activation of HIV-LTR by Tat was significantly blocked by co-transfection with the plasmid expressing antisense RNA against Tat and Rev as observed by transient CAT assays. We then selected MOLT4 cells stably transfected with the Tat-antisense RNA plasmid for further biological studies. However, the substantial evidence has not yet been obtained since viabilities of these cell clones were not good enough to demonstrate the actual remistence to HIV infection.In addition to the above experiments, construction of a retrovirus vector which is able to endow cells with ability to produce antisense RNA to the HIV Tat (and Rev) is now underway. Since this retrovirus vector contains neomycin-resistance gene as well, selection and establishment of those cells producing the antisense RNA has now become much easier. We are attempting to test eagerly if these cells are resistant to HIV infection.