Emiliani S, Van Lint C, Fischle W, Paras P, Ott M, Brady J, Verdin E
The Picower Institute for Medical Research, Manhasset, NY 11030, USA.
Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6377-81. doi: 10.1073/pnas.93.13.6377.
Study of the mechanism of HIV-1 postintegration latency in the ACH2 cell line demonstrates that these cells failed to increase HIV-1 production following treatment with exogenous Tat. Reasoning that the defect in ACH2 cells involves the Tat response, we analyzed the sequence of tat cDNA and Tat responsive element (TAR) from the virus integrated in ACH2. Tat cDNA sequence is closely related to that of HIV LAI, and the encoded protein is fully functional in terms of long terminal repeat (LTR) transactivation. Cloning of a region corresponding to the 5'-LTR from ACH2, however, identified a point mutation (C37 -> T) in TAR. This mutation impaired Tat responsiveness of the LTR in transient transfection assays, and the measured defect was complemented in cells that had been treated with tetradecanoyl phorbol acetate or tumor necrosis factor type alpha (TNF-alpha). A compensatory mutation in TAR (G28 -> A), designed to reestablish base pairing in the TAR hairpin, restored wild-type Tat responsiveness. When the (C37 -> T) mutation was introduced in an infectious clone of HIV-1, no viral production was measured in the absence of TNF-alpha, whereas full complementation was observed when the infection was conducted in the presence of TNF-alpha or when a compensatory mutation (G28 -> A) was introduced into TAR. These experiments identify a novel mutation associated with HIV-1 latency and suggest that alterations in the Tat-TAR axis can be a crucial determinant of the latent phenotype in infected individuals.
对ACH2细胞系中HIV-1整合后潜伏期机制的研究表明，在用外源性Tat处理后，这些细胞未能增加HIV-1的产生。鉴于ACH2细胞中的缺陷涉及Tat反应，我们分析了整合于ACH2中的病毒的tat cDNA和Tat反应元件（TAR）的序列。Tat cDNA序列与HIV LAI的序列密切相关，并且从长末端重复序列（LTR）反式激活方面来看，所编码的蛋白质具有完全功能。然而，从ACH2中克隆对应于5'-LTR的区域时，在TAR中鉴定出一个点突变（C37 -> T）。在瞬时转染试验中，该突变损害了LTR的Tat反应性，并且在用十四烷酰佛波醇乙酸酯或α型肿瘤坏死因子（TNF-α）处理的细胞中，所测得的缺陷得到了补充。在TAR中设计的一个补偿性突变（G28 -> A），旨在重新建立TAR发夹中的碱基配对，恢复了野生型Tat反应性。当在HIV-1的感染性克隆中引入（C37 -> T）突变时，在不存在TNF-α的情况下未检测到病毒产生，而当在TNF-α存在的情况下进行感染或当将补偿性突变（G28 -> A）引入TAR时，则观察到完全互补。这些实验鉴定出了一个与HIV-1潜伏期相关的新突变，并表明Tat-TAR轴的改变可能是感染个体中潜伏表型的关键决定因素。
