Kaposi's Sarcoma-Associated Herpesvirus ori-Lyt-Dependent DNA Replication: cis-Acting Requirements for Replication and ori-Lyt-Associated RNA Transcription
Kaposi's Sarcoma-Associated Herpesvirus ori-Lyt-Dependent DNA Replication: cis-Acting Requirements for Replication and ori-Lyt-Associated RNA Transcription
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DOI：
10.1128/jvi.78.16.8615-8629.2004
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发表时间：
2004-08
影响因子：
5.4
通讯作者：
Yan Wang;Hong Li;Man Yee Chan;F. Zhu;D. Lukac;Yan Yuan
中科院分区：
文献类型：
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作者：
Yan Wang;Hong Li;Man Yee Chan;F. Zhu;D. Lukac;Yan Yuan
疱疹病毒裂解性DNA复制需要顺式作用元件（起点）和反式作用因子（如病毒编码的起点结合蛋白和DNA复制酶）。最近，卡波西肉瘤相关疱疹病毒（KSHV）中的裂解DNA复制起点（ori-Lyt）已被鉴定，并且病毒编码的bZip蛋白K8已被证明特异性结合该起点。为了绘制KSHV ori-Lyt内DNA复制功能所需的顺式作用元件，并确定K8 bZip蛋白与起点结合的性质，我们在KSHV ori-Lyt的核心结构域上构建了连续的内部缺失突变，并在瞬时复制试验中测试了它们的DNA复制功能。该诱变研究允许鉴定ori-Lyt内的四种组分，并且它们都是ori-Lyt功能不可或缺的。第一个组分含有八个CCAAT/增强子结合蛋白（C/EBP）结合基序，其组织为四个间隔的C/EBP回文。每个回文含有两个头对头CCAAT共有基序，它们被13或12 bp的间隔序列分开。这些C/EBP基序的取代诱变表明，这些C/EBP回文都需要K8结合和ori-Lyt依赖的DNA复制。第二个组成部分是一个18 bp的AT回文，这是ori-Lyt功能所必需的。第三个组成部分被确定为一个32 bp的先前未鉴定的序列，是DNA复制所需的。最后一个组件由开放阅读框50（ORF 50）/Rta响应元件（RRE）和TATA盒组成。我们表明，结合的ORF 50/Rta蛋白的RRE是必不可少的ori-Lyt依赖的DNA复制。功能性RRE和下游TATA盒的存在表明该区域作为ORF 50/Rta依赖性启动子，并且转录事件可能是ori-Lyt依赖性DNA复制所必需的。使用荧光素酶报告系统，我们证明了该区域的RRE和TATA盒构成了一个ORF 50/RTA依赖的启动子。此外，在启动子下游鉴定出1.4kb的多腺苷酸化RNA。
ABSTRACT Herpesvirus lytic DNA replication requires both the cis-acting element, the origin, and trans-acting factors such as virally encoded origin-binding protein and DNA replication enzymes. Recently, the origins of lytic DNA replication (ori-Lyt) in Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified and a virally encoded bZip protein, K8, has been shown to specifically bind to the origin. To map cis-acting elements within KSHV ori-Lyt that are required for DNA replication function and to define the nature of K8 bZip protein binding to the origin, we constructed consecutive internal deletion mutations across the core domain of a KSHV ori-Lyt and tested them for DNA replication function in a transient replication assay. This mutagenesis study allowed the identification of four components within the ori-Lyt, and all were indispensable for ori-Lyt function. The first component contains eight CCAAT/enhancer binding protein (C/EBP) binding motifs that organize as four spaced C/EBP palindromes. Each palindrome contains two head-to-head CCAAT consensus motifs that are separated by a 13- or 12-bp space sequence. Substitution mutagenesis of these C/EBP motifs showed that these C/EBP palindromes are required for both K8 binding and ori-Lyt-dependent DNA replication. The second component is an 18-bp AT palindrome, which is essential for ori-Lyt function. The third component was determined to be a 32-bp previously unidentified sequence and is required for DNA replication. The last component consists of an open reading frame 50 (ORF50)/Rta responsive element (RRE) and a TATA box. We showed that the binding of an ORF50/Rta protein to the RRE was essential for ori-Lyt-dependent DNA replication. The presence of a functional RRE and a downstream TATA box suggested that this region serves as an ORF50/Rta-dependent promoter and a transcription event may be necessary for ori-Lyt-dependent DNA replication. Using a luciferase reporter system, we demonstrated that the region of the RRE and TATA box constitutes an ORF50/Rta-dependent promoter. Furthermore, a polyadenylated RNA of 1.4 kb was identified downstream of the promoter.