Genome-wide identification of binding sites for Kaposi's sarcoma-associated herpesvirus lytic switch protein, RTA.
Genome-wide identification of binding sites for Kaposi's sarcoma-associated herpesvirus lytic switch protein, RTA.
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DOI：
10.1016/j.virol.2009.01.031
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发表时间：
2009-04-10
期刊：
影响因子：
3.7
通讯作者：
Gao, Shou-Jiang
中科院分区：
文献类型：
--
作者：
Chen, Jiguo;Ye, Fengchun;Xie, Jianping;Kuhne, Kurt;Gao, Shou-Jiang
关键词：
Kaposi肉瘤相关疱疹病毒(KSHV)复制和转录激活因子(RTA)由ORF50编码，是一种裂解开关蛋白，用于病毒从潜伏期重新激活。RTA的表达激活了下游病毒基因的表达，是启动完整的病毒裂解程序所必需的。利用染色质免疫沉淀结合KSHV全基因组芯片技术，我们在KSHV感染细胞系BCBL-1中鉴定了19个RTA结合位点。这些结合位点位于KSHV基因的启动子、内含子或外显子区域，包括ORF8、ORFK4.1、ORFK5、PAN、ORF16、ORF29、ORF45、ORF50、ORFK8、ORFK10.1、ORF59、ORFK12、ORF71/72、ORFK14/ORF74和ORFK15，以及MicroRNA簇。我们通过芯片和实时定量聚合酶链式反应证实了这些RTA结合位点的存在。我们进一步定位了ORFK15基因第一内含子中的RTA结合位点，并确定它是RTA反应的。ORFK15 RTA结合序列TTCCAGGAA TTCCTGGAA由两个串联重复序列的回文结构组成，每个串联重复序列本身也是一个不完美的反向重复序列。报告实验和凝胶迁移率改变实验证实RTA蛋白在体外与该序列结合。与其他RTA结合位点的序列比对表明，RTA共识结合基序为TTCCAGGAT(N)0-16TTCCTGGGA。有趣的是，大多数已鉴定的RTA结合位点只包含该RTA结合基序的一半或部分。这些结果提示RTA在体内结合的复杂性，以及在RTA反式激活靶基因的过程中其他细胞或病毒转录因子的参与。
Kaposi’s sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) encoded by ORF50 is a lytic switch protein for viral reactivation from latency. The expression of RTA activates the expression of downstream viral genes, and is necessary for triggering the full viral lytic program. Using chromatin immunoprecipitation assay coupled with a KSHV whole-genome tiling microarray (ChIP-on-chip) approach, we identified a set of 19 RTA binding sites in the KSHV genome in a KSHV-infected cell line BCBL-1. These binding sites are located in the regions of promoters, introns, or exons of KSHV genes including ORF8, ORFK4.1, ORFK5, PAN, ORF16, ORF29, ORF45, ORF50, ORFK8, ORFK10.1, ORF59, ORFK12, ORF71/72, ORFK14/ORF74, and ORFK15, the two origins of lytic replication OriLyt-L and OriLyt-R, and the microRNA cluster. We confirmed these RTA binding sites by ChIP and quantitative real-time PCR. We further mapped the RTA binding site in the first intron of the ORFK15 gene, and determined that it is RTA-responsive. The ORFK15 RTA binding sequence TTCCAGGAA TTCCTGGAA consists of a palindromic structure of two tandem repeats, of which each itself is also an imperfect inverted repeat. Reporter assay and electrophoretic mobility shift assay confirmed the binding of the RTA protein to this sequence in vitro. Sequence alignment with other RTA binding sites identified the RTA consensus binding motif as TTCCAGGAT(N)0–16TTCCTGGGA. Interestingly, most of the identified RTA binding sites contain only half or part of this RTA binding motif. These results suggest the complexity of RTA binding in vivo, and the involvements of other cellular or viral transcription factors during RTA transactivation of target genes.
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