Ambrosino C, Ruocco M R, Chen X, Mallardo M, Baudi F, Trematerra S, Quinto I, Venuta S, Scala G
Department of Clinical and Experimental Medicine, Medical School, University of Reggio Calabria, 88100 Catanzaro, Italy.
J Biol Chem. 1997 Jun 6;272(23):14883-92. doi: 10.1074/jbc.272.23.14883.
Human immunodeficiency virus type 1 (HIV-1) infection is associated with severe psoriasis, B cell lymphoma, and Kaposi's sarcoma. A deregulated production of interleukin-6 (IL6) has been implicated in the pathogenesis of these diseases. The molecular mechanisms underlying the abnormal IL6 secretion of HIV-1-infected cells may include transactivation of the IL6 gene by HIV-1. Here we report the molecular mechanisms of Tat activity on the expression of the IL6 gene. By using 5' deletion mutants of pIL6Pr-CAT and using IL6:HIV-1-LTR hybrid constructs where discrete regions of the IL6 promoter replaced the TAR sequence in HIV-1 LTR, we identified a short sequence of the 5'-untranslated region of the IL6 mRNA that is required for Tat to trans-activate the IL6 promoter. This sequence acquires a stem-loop structure and includes a UCU sequence that binds to Tat and is necessary for full trans-activation. In addition, we provide the evidence that Tat can function by enhancing the CAAT enhancer-binding protein (C/EBP) DNA binding activity and is able to complex with in vitro translated C/EBPbeta, which is a major mediator of IL6 promoter function. By using the yeast two-hybrid system and immunoprecipitation, we observed that the interaction of Tat with C/EBP proteins also occurred in vivo. The data are consistent with the possibility that Tat may function on heterologous genes by interacting with RNA structures possibly present in a large number of cellular and viral genes. In addition, Tat may function by protein-protein interactions, leading to the generation of heterodimers with specific transcription factors.
1型人类免疫缺陷病毒(HIV-1)感染与严重银屑病、B细胞淋巴瘤和卡波西肉瘤有关。白细胞介素-6(IL6)的失调产生与这些疾病的发病机制有关。HIV-1感染细胞异常分泌IL6的分子机制可能包括HIV-1对IL6基因的反式激活。在此,我们报告Tat活性对IL6基因表达的分子机制。通过使用pIL6Pr-CAT的5'缺失突变体,并使用IL6:HIV-1-LTR杂交构建体,其中IL6启动子的离散区域取代了HIV-1 LTR中的TAR序列,我们鉴定出IL6 mRNA 5'-非翻译区的一段短序列,这是Tat反式激活IL6启动子所必需的。该序列形成茎环结构,并包含一个与Tat结合的UCU序列,这是完全反式激活所必需的。此外,我们提供证据表明,Tat可以通过增强CAAT增强子结合蛋白(C/EBP)的DNA结合活性发挥作用,并且能够与体外翻译的C/EBPβ形成复合物,C/EBPβ是IL6启动子功能的主要调节因子。通过使用酵母双杂交系统和免疫沉淀,我们观察到Tat与C/EBP蛋白的相互作用也发生在体内。这些数据与Tat可能通过与大量细胞和病毒基因中可能存在的RNA结构相互作用而作用于异源基因的可能性一致。此外,Tat可能通过蛋白质-蛋白质相互作用发挥作用,导致与特定转录因子形成异二聚体。