Panagoulias Ioannis, Karagiannis Fotios, Aggeletopoulou Ioanna, Georgakopoulos Tassos, Argyropoulos Christos P, Akinosoglou Karolina, Gogos Charalambos, Skoutelis Athanasios, Mouzaki Athanasia
Division of Hematology, Department of Internal Medicine, Medical School, University of Patras, Patras, Greece.
Division of Nephrology, Department of Internal Medicine, Medical School, University of New Mexico, Albuquerque, NM, United States.
Front Immunol. 2018 Jan 4;8:1924. doi: 10.3389/fimmu.2017.01924. eCollection 2017.
HIV-1 is transcriptionally active in activated T helper (Th)-cells and inactive in naive or resting memory Th-cells. Ets-2 is a preinduction transcriptional repressor of the IL-2 gene in naive Th-cells and a candidate transcriptional repressor of HIV-1 in the same cells, because the -279 to -250 upstream region of HIV-1-LTR [repressor-activator target sequence (RATS)], that participates in HIV-1-LTR transcriptional silencing, encompasses the AAGGAG Ets-2 binding site. In this proof of concept study, we investigated whether Ets-2 represses the expression of HIV-1. To assess whether Ets-2 can repress HIV-1 transcriptional activation acting through RATS, we transfected Jurkat cells with an Ets-2 overexpression plasmid (pCDNA3-ets-2) or Ets-2 silencing plasmids (ets-2-shRNA) and, as target genes, plasmids carrying the whole HIV-1-LTR sequence (HIV-1-LTR-CAT) or two copies of the RATS sequence (2× RATS-CAT) or a point mutation in the Ets-2 binding site (2× mutantRATS-CAT) or CMV-CAT (control). Ets-2 overexpression resulted in a significant reduction of HIV-1-LTR-CAT and 2× RATS-CAT activities in stimulated cells, but not of the 2× mutantRATS-CAT or CMV-CAT. Ets-2 silencing led to increased activities of HIV-1-LTR-CAT and 2× RATS-CAT in unstimulated cells, but had no effect on the activities of 2× mutantRATS-CAT and CMV-CAT. To assess Ets-2 binding to HIV-1-LTR-RATS in naive Th-cells, we isolated naive Th-cell nuclear proteins and passed them through an Ets-2 antibody column; electrophoretic mobility shift assays were performed using an RATS probe mixed with consecutive protein eluates. Ets-2 bound to the HIV-1-LTR-RATS in a dose-dependent manner. To assess Ets-2 binding to RATS , Jurkat cells were transfected with 2× RATS-CAT and stained for the Ets-2 protein and the RATS sequence by combining immunofluorescence and fluorescence hybridization techniques. In unstimulated cells, Ets-2 bound to RATS, whereas no binding was observed in stimulated cells. To test for RATS specificity, the same experiments were performed with 2× mutantRATS-CAT, and no binding of Ets-2 was observed. The results were corroborated by chromatin immunoprecipitation assays performed with the same cells. Our results show that Ets-2 is a transcriptional repressor of HIV-1. Repression of HIV-LTR-RATS mediated by Ets-2 may account for the low-level transcription and replication of HIV-1 in naive Th-cells, and contribute to the viral latency and maintenance of viral reservoirs in patients, despite long-term therapy.
HIV-1在活化的辅助性T（Th）细胞中具有转录活性，而在初始或静息记忆Th细胞中无转录活性。Ets-2是初始Th细胞中白细胞介素-2基因的诱导前转录抑制因子，也是同一细胞中HIV-1的候选转录抑制因子，因为参与HIV-1长末端重复序列（LTR）转录沉默的HIV-1-LTR上游-279至-250区域[抑制因子-激活因子靶序列（RATS）]包含AAGGAG Ets-2结合位点。在这项概念验证研究中，我们调查了Ets-2是否抑制HIV-1的表达。为评估Ets-2是否能通过RATS抑制HIV-1转录激活，我们用Ets-2过表达质粒（pCDNA3-ets-2）或Ets-2沉默质粒（ets-2-shRNA）转染Jurkat细胞，并将携带完整HIV-1-LTR序列的质粒（HIV-1-LTR-CAT）或两份RATS序列的质粒（2×RATS-CAT）或Ets-2结合位点的点突变质粒（2×mutantRATS-CAT）或CMV-CAT（对照）作为靶基因。Ets-2过表达导致刺激细胞中HIV-1-LTR-CAT和2×RATS-CAT活性显著降低，但2×mutantRATS-CAT或CMV-CAT活性未降低。Ets-2沉默导致未刺激细胞中HIV-1-LTR-CAT和2×RATS-CAT活性增加，但对2×mutantRATS-CAT和CMV-CAT活性无影响。为评估Ets-2与初始Th细胞中HIV-1-LTR-RATS的结合，我们分离了初始Th细胞核蛋白并使其通过Ets-2抗体柱；使用与连续蛋白洗脱液混合的RATS探针进行电泳迁移率变动分析。Ets-2以剂量依赖方式与HIV-1-LTR-RATS结合。为评估Ets-2与RATS的结合，用2×RATS-CAT转染Jurkat细胞，并通过免疫荧光和荧光杂交技术结合对Ets-2蛋白和RATS序列进行染色。在未刺激细胞中，Ets-2与RATS结合，而在刺激细胞中未观察到结合。为测试RATS特异性，对2×mutantRATS-CAT进行相同实验，未观察到Ets-2结合。相同细胞进行的染色质免疫沉淀分析证实了结果。我们的结果表明Ets-2是HIV-1的转录抑制因子。Ets-2介导的对HIV-LTR-RATS的抑制可能解释了HIV-1在初始Th细胞中的低水平转录和复制，并有助于患者体内病毒潜伏和病毒储存库的维持，尽管进行了长期治疗。
