Gyotoku J, el-Farrash M A, Fujimoto S, Germeraad W T, Watanabe Y, Teshigawara K, Harada S, Katsura Y
Department of Immunology, Kyoto University, Japan.
Virus Genes. 1991 Jul;5(3):189-202. doi: 10.1007/BF00568969.
The effect of the expression of antisense RNA against the human immunodeficiency virus (HIV) genome in a human T-cell line CEM on HIV replication was investigated. A 2.7 kilobase (kb) fragment of the HIV genome, including tat and a part of rev and env, was cloned into the retroviral vector WB in the antisense orientation under the SV40 or H-2K promoter. CEM cells transduced with this antisense gene via recombinant retrovirus expressed the RNAs of three different molecular sizes containing the antisense construct. CEM cells and these transduced cells were infected with HIV. HIV replication was evaluated 4-10 days later by an immunofluorescence assay and by determining the reverse transcriptase activity in the culture supernatant. The results indicate that although the recombinant retrovirus WB strongly enhanced the HIV replication in CEM cells, the expression of antisense RNA in the cells was highly effective in impeding the replication of HIV. The inhibitory effect was especially high in CEM cells transduced with the antisense gene under the control of SV40 promoter. In this case, HIV antigen-positive cells and reverse transcriptase activity in the culture supernatant of transduced cells were reduced to 30-50% and 5-10% of those in CEM cells and in the CEM cells transduced with WB, respectively.
研究了在人T细胞系CEM中，针对人类免疫缺陷病毒（HIV）基因组的反义RNA表达对HIV复制的影响。将HIV基因组的一个2.7千碱基（kb）片段，包括tat以及rev和env的一部分，以反义方向克隆到逆转录病毒载体WB中，置于SV40或H - 2K启动子的控制下。通过重组逆转录病毒转导了该反义基因的CEM细胞表达了含有反义构建体的三种不同分子大小的RNA。用HIV感染CEM细胞和这些转导细胞。4 - 10天后，通过免疫荧光测定法并通过测定培养上清液中的逆转录酶活性来评估HIV复制。结果表明，尽管重组逆转录病毒WB强烈增强了CEM细胞中的HIV复制，但细胞中反义RNA的表达在阻碍HIV复制方面非常有效。在SV40启动子控制下转导了反义基因的CEM细胞中，抑制作用尤为显著。在这种情况下，转导细胞培养上清液中的HIV抗原阳性细胞和逆转录酶活性分别降至CEM细胞以及用WB转导的CEM细胞中的30 - 50%和5 - 10%。
