Zachar V, Ebbesen P, Thomas R A, Zacharova V, Goustin A S
Center for Molecular Biology, Wayne State University, Detroit, Michigan 48202.
J Gen Virol. 1994 Jun;75 ( Pt 6):1461-8. doi: 10.1099/0022-1317-75-6-1461.
We have analysed the capacity of the trophoblast-derived malignant cell lines BeWo, JAR and JEG-3, and primary cultures of highly purified trophoblast cells to support the basal and Tat-mediated trans-activation-enhanced transcriptional activity of two distinct human immunodeficiency virus type 1 (HIV-1) isolates. Kinetic studies based on expression of long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) constructs revealed that LTRs of both the prototype strain 3B and the highly cytopathic Zairean variant NDK were activated significantly in all target cells. Overall, the strongest activation was observed in primary trophoblasts. A novel modification of quantitative PCR was used to normalize LTR expression for transfection efficiency, enabling the calculation of specific expression rates in terms of muU CAT enzyme per fmol of transfected DNA. Using the latter criterion we determined that LTRs of both viruses were activated in decreasing order from trophoblasts to JAR, JEG-3 and BeWo cells; furthermore, the expression of HIV-1 3B LTR always significantly surpassed that of HIV-1 NDK. The effects of trans-activation on either of the LTRs, when assayed in cotransfection assays with various amounts of HIV-1 NDK-Tat expression vector, increased in a dose-dependent fashion and were comparable in a particular neoplastic cell line. Furthermore, the cell-specific LTR activity patterns did not correspond to the abundance of transcription factors binding specifically to the viral NF kappa B and SP1 motifs. Unlike SP1-binding proteins which were relatively abundant, substantially smaller amounts of proteins with NF kappa B specificity were found in all cells. Despite this apparent deficit in NF kappa B activity, trophoblasts supported a high basal activity of both LTRs. These data indicate that an insufficiency of basal or Tat-trans-activated LTR activity cannot account for the low level of HIV-1 replication in this important cell type.
我们分析了滋养层来源的恶性细胞系BeWo、JAR和JEG-3，以及高度纯化的滋养层细胞原代培养物，以支持两种不同的人类免疫缺陷病毒1型（HIV-1）分离株的基础转录活性和Tat介导的反式激活增强的转录活性。基于长末端重复序列（LTR）-氯霉素乙酰转移酶（CAT）构建体表达的动力学研究表明，原型毒株3B和高细胞病变性的扎伊尔变种NDK的LTR在所有靶细胞中均被显著激活。总体而言，在原代滋养层细胞中观察到最强的激活。一种定量PCR的新方法被用于将LTR表达标准化以衡量转染效率，从而能够计算每飞摩尔转染DNA的毫微单位CAT酶的特异性表达率。根据后一标准，我们确定两种病毒的LTR从滋养层细胞到JAR、JEG-3和BeWo细胞的激活程度依次降低；此外，HIV-1 3B LTR的表达总是显著超过HIV-1 NDK。当用不同量的HIV-1 NDK-Tat表达载体在共转染实验中检测时，反式激活对任一LTR的影响呈剂量依赖性增加，并且在特定的肿瘤细胞系中具有可比性。此外，细胞特异性的LTR活性模式与特异性结合病毒NFκB和SP1基序的转录因子丰度不对应。与相对丰富的SP1结合蛋白不同，在所有细胞中发现具有NFκB特异性的蛋白量要少得多。尽管NFκB活性存在明显不足，但滋养层细胞支持两种LTR的高基础活性。这些数据表明，基础或Tat反式激活的LTR活性不足不能解释HIV-1在这种重要细胞类型中的低水平复制。
