Martin-Serrano Juan, Li Kelvin, Bieniasz Paul D
Aaron Diamond AIDS Research Center, Rockefeller University, New York, New York 10016, USA.
J Virol. 2002 Jan;76(1):208-19. doi: 10.1128/jvi.76.1.208-219.2002.
Cyclin T1 (CycT1), a component of positive-transcription-elongation factor-b (P-TEFb), is an essential cofactor for transcriptional activation by lentivirus Tat proteins. It is thought that low CycT1 expression levels restrict human immunodeficiency virus type 1 (HIV-1) expression levels and replication in resting CD4+ lymphocytes. In this study, we undertook a functional analysis of the cycT1 promoter to determine which, if any, promoter elements might be responsible for cellular activation state-dependent CycT1 expression. The cycT1 gene contains a complex promoter that exhibits an extreme degree of functional redundancy: five nonoverlapping fragments were found to exhibit significant promoter activity in immortalized cell lines, and these elements could interact in a synergistic or redundant manner to mediate cycT1 transcription. Reporter gene expression, mediated by the cycT1 promoter, was detectable in unstimulated transfected primary lymphocytes and multiple sites within the promoter could serve to initiate transcription. While utilization of these start sites was significantly altered by the application of exogenous stimuli to primary lymphocytes and two distinct promoter elements exhibited enhanced activity in the presence of phorbol ester, overall cycT1 transcription was only modestly enhanced in response to cell activation. These observations prompted a reexamination of CycT1 protein expression in primary lymphocytes. In fact, steady-state CycT1 expression is only slightly lower in unstimulated lymphocytes compared to phorbol ester-treated cells or a panel of immortalized cell lines. Importantly, CycT1 is expressed at sufficient levels in unstimulated primary cells to support robust Tat activity. These results strongly suggest that CycT1 expression levels in unstimulated primary lymphocytes do not profoundly limit HIV-1 gene expression or provide an adequate mechanistic explanation for proviral latency in vivo.
细胞周期蛋白T1（CycT1）是正转录延伸因子b（P-TEFb）的一个组成部分，是慢病毒Tat蛋白转录激活所必需的辅助因子。据认为，低水平的CycT1表达会限制1型人类免疫缺陷病毒（HIV-1）在静息CD4+淋巴细胞中的表达水平和复制。在本研究中，我们对cycT1启动子进行了功能分析，以确定哪些启动子元件（如果有的话）可能负责细胞激活状态依赖性的CycT1表达。cycT1基因包含一个复杂的启动子，该启动子表现出极高程度的功能冗余：在永生化细胞系中发现五个不重叠的片段具有显著的启动子活性，并且这些元件可以以协同或冗余的方式相互作用以介导cycT1转录。由cycT1启动子介导的报告基因表达在未刺激的转染原代淋巴细胞中可检测到，并且启动子内的多个位点可用于启动转录。虽然对原代淋巴细胞施加外源性刺激会显著改变这些起始位点的利用情况，并且两个不同的启动子元件在佛波酯存在下表现出增强的活性，但总体而言，cycT1转录仅在细胞激活时适度增强。这些观察结果促使我们重新审视原代淋巴细胞中CycT1蛋白的表达。事实上，与佛波酯处理的细胞或一组永生化细胞系相比，未刺激的淋巴细胞中CycT1的稳态表达仅略低。重要的是，CycT1在未刺激的原代细胞中表达水平足以支持强大的Tat活性。这些结果强烈表明，未刺激的原代淋巴细胞中CycT1的表达水平不会深刻限制HIV-1基因表达，也不能为体内前病毒潜伏提供充分的机制解释。
