Transcription activation of polyadenylated nuclear RNA by Rta in human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus
Transcription activation of polyadenylated nuclear RNA by Rta in human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus
复制标题
DOI：
10.1128/jvi.75.7.3129-3140.2001
复制
发表时间：
2001-04-01
影响因子：
5.4
通讯作者：
Sun, R
中科院分区：
文献类型：
--
作者：
Song, MJ;Brown, HJ;Sun, R
人类疱疹病毒8（HHV-8）（也称为卡波西肉瘤相关疱疹病毒）在裂解复制的早期阶段编码一种新的非编码多聚腺苷酸化核（PAN）RNA（也称为T1.1或nut-1）。PAN RNA是HHV-8最丰富的转录物，在裂解复制期间，在HHV-8感染的细胞中占总poly（A）选择性转录物的80%。我们通过在溴化乙锭染色的凝胶中观察来自poly（A）选择的RNA的1.1- 1.2- kb PAN RNA来直接测量PAN RNA的丰度。我们进一步研究了PAN RNA表达被诱导到如此高水平的机制，rta是HHV-8的立即早期基因，是激活潜伏感染细胞中裂解基因表达的充分和必要的反式激活因子。先前显示Rta的异位表达诱导来自内源性病毒基因组的PAN RNA表达并激活报告系统中的PAN启动子。在这里，我们已经鉴定了PAN启动子中的Rta响应元件（RRE）。缺失分析显示，RRE存在于PAN启动子的核苷酸-69和-38之间的区域中。含有PAN启动子的转录起始位点上游的69个核苷酸的启动子构建体在不存在或存在HHV-8基因组的情况下被Rta激活。Rta在293 T细胞中激活PAN启动子高达7，000倍，在B细胞中激活2，000倍。电泳迁移率变动分析表明，Rta与PAN启动子的RRE形成高度稳定的复合物。我们的研究表明，Rta可以诱导PAN RNA的表达，通过直接结合Rta的PAN启动子的RRE。这项研究突出了一个重要的机制控制PAN RNA的表达，也提供了一个模型系统，研究如何Rta反式激活基因表达裂解复制过程中。
Human herpesvirus 8 (HHV-8) (also known as Kaposi's sarcoma-associated herpesvirus) encodes a novel noncoding polyadenylated nuclear (PAN) RNA (also known as T1.1 or nut-1) during the early phase of lytic replication. PAN RNA is the most abundant transcript of HHV-8, comprising 80% of total poly(A)-selected transcripts in HHV-8-infected cells during lytic replication. We directly measured the abundance of PAN RNA by visualizing 1.1- to 1.2- kb PAN RNA in an ethidium bromide-stained gel from poly(A)-selected RNA. We further pursued the mechanisms by which PAN RNA expression is induced to such high levels, rta, an immediate-early gene of HHV-8, is a transactivator that is sufficient and necessary to activate lytic gene expression in latently infected cells. Ectopic expression of Rta was previously shown to induce PAN RNA expression from the endogenous viral genome and activate the PAN promoter in a reporter system. Here, we have identified the Rta-responsive element (RRE) in the PAN promoter. Deletion analysis revealed that the RRE is present in a region between nucleotides -69 and -38 of the PAN promoter. A promoter construct containing the 69 nucleotides upstream of the transcription start site of the PAN promoter was activated by Rta in the absence or presence of the HHV-8 genome. Rta activated the PAN promoter up to 7,000-fold in 293T cells and 2,000-fold in B cells. Electrophoretic mobility shift assays demonstrated that Rta formed a highly stable complex with the RRE of the PAN promoter. Our study suggests that Rta can induce PAN RNA expression by direct binding of Rta to the RRE of the PAN promoter. This study has highlighted an important mechanism controlling PAN RNA expression and also provides a model system for investigating how Rta transactivates gene expression during lytic replication.