Niederman T M, Hastings W R, Luria S, Bandres J C, Ratner L
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Virology. 1993 May;194(1):338-44. doi: 10.1006/viro.1993.1264.
The human immunodeficiency virus type 1 long terminal repeat, HIV-1-LTR, contains binding sites for several cellular transcription factors which contribute to HIV-1 gene expression. Our previous studies on the function of the HIV-1-encoded Nef protein suggested that Nef may be an inhibitor HIV-1 transcription. To determine whether Nef affects the binding of cellular factors implicated in HIV-1 regulation, 32P-labeled oligonucleotides corresponding to the binding sites were incubated with nuclear extracts prepared from Nef-expressing T-cell lines that were not stimulated or were stimulated with T-cell mitogens. We found that Nef inhibited the recruitment of AP-1 DNA-binding activity in mitogen-stimulated human T-cells. Additionally, Nef expressing cells were transiently transfected with a plasmid in which HIV-1 AP-1 DNA recognition sequences were cloned downstream of the chloramphenicol acetyltransferase (CAT) gene. Mitogen-mediated transcriptional activation of the CAT gene in this construct was inhibited in Nef-expressing cells but not in control cells. These studies suggest that, by inhibiting AP-1 activation, Nef may play a role in regulating HIV-1 gene expression in infected T-cells.
1型人类免疫缺陷病毒长末端重复序列（HIV-1-LTR）含有多个细胞转录因子的结合位点，这些因子有助于HIV-1基因表达。我们之前对HIV-1编码的Nef蛋白功能的研究表明，Nef可能是HIV-1转录的抑制剂。为了确定Nef是否影响与HIV-1调控相关的细胞因子的结合，将对应于结合位点的32P标记寡核苷酸与从未受刺激或受T细胞有丝分裂原刺激的表达Nef的T细胞系制备的核提取物一起孵育。我们发现，Nef抑制有丝分裂原刺激的人T细胞中AP-1 DNA结合活性的募集。此外，用一个质粒瞬时转染表达Nef的细胞，该质粒中HIV-1 AP-1 DNA识别序列克隆在氯霉素乙酰转移酶（CAT）基因的下游。在表达Nef的细胞中，该构建体中CAT基因的有丝分裂原介导的转录激活受到抑制，而在对照细胞中则未受抑制。这些研究表明，通过抑制AP-1激活，Nef可能在调节受感染T细胞中的HIV-1基因表达中发挥作用。
