Song Moon Jung, Deng Hongyu, Sun Ren
Department of Molecular and Medical Pharmacology, UCLA AIDS Institute, Jonsson Comprehensive Cancer Center, and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095, USA.
J Virol. 2003 Sep;77(17):9451-62. doi: 10.1128/jvi.77.17.9451-9462.2003.
Replication and transcription activator (RTA) (also referred to as ORF50), an immediate-early gene product of Kaposi's sarcoma-associated herpesvirus (KSHV)/(human herpesvirus 8), plays a critical role in balancing the viral life cycle between latency and lytic replication. RTA has been shown to act as a strong transcription activator for several downstream genes of KSHV. Direct binding of RTA to DNA is thought to be one of the important mechanisms for transactivation of target genes, while indirect mechanisms are also implicated in RTA transactivation of certain selected genes. This study demonstrated direct binding of the DNA-binding domain of RTA (Rdbd) to a Kaposin (Kpsn) promoter sequence, which is highly homologous to the RTA-responsive element (RRE) of the PAN promoter. We undertook a comparative study of the RREs of PAN RNA, ORF57, vIL-6, and Kpsn to understand how RTA regulates gene expression during lytic replication. Comparing RNA abundance and transcription initiation rates of these RTA target genes in virus-infected cells suggested that the transcription initiation rate of the promoters is a major determinant of viral gene expression, rather than stability of the transcripts. RTA-mediated transactivation of reporters containing each RRE showed that their promoter strengths in a transient-transfection system were comparable to their transcription rates during reactivation. Moreover, our electrophoretic mobility shift assays of each RRE demonstrated that the highly purified Rdbd protein directly bound to the RREs. Based on these results, we conclude that direct binding of RTA to these target sequences contributes to their gene expression to various extents during the lytic life cycle of KSHV.
复制和转录激活因子（RTA）（也称为ORF50）是卡波西肉瘤相关疱疹病毒（KSHV）/（人类疱疹病毒8）的一种立即早期基因产物，在平衡病毒潜伏和裂解复制的生命周期中起着关键作用。RTA已被证明是KSHV几个下游基因的强转录激活因子。RTA与DNA的直接结合被认为是靶基因反式激活的重要机制之一，而间接机制也与RTA对某些选定基因的反式激活有关。本研究证明了RTA的DNA结合结构域（Rdbd）与卡波辛（Kpsn）启动子序列的直接结合，该序列与PAN启动子的RTA反应元件（RRE）高度同源。我们对PAN RNA、ORF57、vIL-6和Kpsn的RRE进行了比较研究，以了解RTA在裂解复制过程中如何调节基因表达。比较病毒感染细胞中这些RTA靶基因的RNA丰度和转录起始率表明，启动子的转录起始率是病毒基因表达的主要决定因素，而不是转录本的稳定性。RTA介导的含有每个RRE的报告基因的反式激活表明，它们在瞬时转染系统中的启动子强度与再激活期间的转录率相当。此外，我们对每个RRE的电泳迁移率变动分析表明，高度纯化的Rdbd蛋白直接与RRE结合。基于这些结果，我们得出结论，在KSHV的裂解生命周期中，RTA与这些靶序列的直接结合在不同程度上促进了它们的基因表达。
