Autoregulation of DNA binding and protein stability of Kaposi's sarcoma-associated herpesvirus ORF50 protein
Autoregulation of DNA binding and protein stability of Kaposi's sarcoma-associated herpesvirus ORF50 protein
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DOI：
10.1128/jvi.78.19.10657-10673.2004
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发表时间：
2004-10-01
影响因子：
5.4
通讯作者：
Miller, G
中科院分区：
文献类型：
--
作者：
Chang, PJ;Miller, G
在卡波西肉瘤相关疱疹病毒（KSHV）开放阅读框50 （ORF50）中编码的转录激活子启动病毒裂解周期。ORF50蛋白通过至少两种机制激活下游KSHV靶基因：直接识别启动子DNA中的应答元件和与启动子DNA结合的细胞蛋白相互作用。我们已经确定了一个多功能调控区域，存在于ORF50蛋白的氨基酸（aa） 520至535中，控制DNA结合和蛋白质稳定性。电泳迁移率变化、DNA亲和层析和染色质免疫沉淀分析显示，ORF50调控区aa 521至534的缺失或一个基本基序（KKRK）的突变显著增强了ORF50蛋白与DNA的结合。调控区域的缺失和KKRK基序的突变也导致电泳迁移性变异ORF50B的大量表达，ORF50B似乎是ORF50蛋白的一种形式，在翻译后修饰中减少。ORF50B的DNA结合增强和表达增强是相互独立的现象。该调控区可能通过与aa I至390的DNA结合域相互作用抑制DNA结合，并通过与aa 590至650的结构域相互作用使ORF50B不稳定。KKRK基序的突变体在DNA结合中增强，但在激活直接靶标（如聚腺苷化核RNA）和间接靶标（如ORF50本身）时受损。自调节区域的鉴定强调了ORF50蛋白的许多功能必须受到精细的控制，以实现最佳的KSHV裂解周期基因表达。
A transcriptional activator encoded in open reading frame 50 (ORF50) of Kaposi's sarcoma-associated herpesvirus (KSHV) initiates the viral lytic cycle. ORF50 protein activates downstream KSHV target genes by at least two mechanisms: direct recognition of response elements in promoter DNA and interaction with cellular proteins bound to promoter DNA. We have identified a multifunctional regulatory region, present in amino acids (aa) 520 to 535 of ORF50 protein, that controls DNA binding and protein stability. Deletion of aa 521 to 534 or mutation of a basic motif (KKRK) in this regulatory region dramatically enhances DNA binding by ORF50 protein, as shown by electrophoretic mobility shift, DNA affinity chromatography, and chromatin immunoprecipitation assays. Deletion of the regulatory region and mutations in the KKRK motif also lead to abundant expression of an electrophoretic mobility variant, ORF50B, which appears to be a form of ORF50 protein that is decreased in posttranslational modification. Enhanced DNA binding and enhanced expression of ORF50B are independent phenomena. The regulatory region likely inhibits DNA binding through interactions with the DNA binding domain in aa I to 390 and destabilizes ORF50B through interactions with a domain located in aa 590 to 650. Mutants in the KKRK motif that are enhanced in DNA binding are nonetheless impaired in activating direct targets, such as polyadenylated nuclear RNA, and indirect targets, such as ORF50 itself. The identification of an autoregullatory region emphasizes that the many functions of ORF50 protein must be subject to exquisite control to achieve optimal KSHV lytic-cycle gene expression.