The unique IR2 protein of equine herpesvirus 1 negatively regulates viral gene expression
The unique IR2 protein of equine herpesvirus 1 negatively regulates viral gene expression
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DOI：
10.1128/jvi.80.10.5041-5049.2006
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发表时间：
2006-05-01
影响因子：
5.4
通讯作者：
O'Callaghan, Dennis J.
中科院分区：
文献类型：
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作者：
Kim, Scong K.;Ahn, Byung C.;O'Callaghan, Dennis J.
IR2 蛋白 (IR2P) 是立即早期蛋白 (IEP) 的截短形式，缺乏必需的酸性转录激活结构域 (TAD) 和富含丝氨酸的区域，但保留了 DNA 和 TFIIB 的结合结构域以及核定位信号 (NLS)。 IR2启动子的分析表明IR2启动子被EICP0P上调。感染后 5 小时，在马疱疹病毒 1 (EM-1) 感染的 HeLa 和马 NBL6 细胞中首次在细胞核中检测到 IR2P。瞬时转染测定表明，(i) IR2P 本身以剂量依赖性方式下调 EHV-1 早期启动子（EICP0、TK、EICP22 和 EICP27）； (ii) IR2P 以剂量依赖性方式消除 IEP 和 EICP27P (UL5) 介导的病毒启动子反式激活，(iii) IR2P 与 IEP 本身一样，也下调 IE 启动子，表明 IEP TAD 不需要下调 IE 启动子。体外相互作用测定表明 IR2P 与 TATA 盒结合蛋白 (TBP) 相互作用。介导负调节的 IR2P 必需结构域被定位到氨基酸残基 1 至 706，表明 IR2P 的 DNA 结合结构域和 NLS 可能对下调很重要。在瞬时转染和病毒生长测定中，与空载体转染细胞中获得的病毒滴度相比，IR2P 将 EHV-1 产量降低了 23 倍。总体而言，这些研究表明，IR2P 通过充当显性失活蛋白来下调病毒基因表达，该蛋白可阻止 IEP 与病毒启动子的结合和/或抑制 TFIIB 和 TBP 的有限供应。
The IR2 protein (IR2P) is a truncated form of the immediate-early protein (IEP) lacking the essential acidic transcriptional activation domain (TAD) and serine-rich tract and yet retaining binding domains for DNA and TFIIB and nuclear localization signal (NLS). Analysis of the IR2 promoter indicated that the IR2 promoter was upregulated by the EICP0P. The IR2P was first detected in the nucleus at 5 h postinfection in equine herpesvirus 1 (EM-1)-infected HeLa and equine NBL6 cells. Transient-transfection assays revealed that (i) the IR2P by itself downregulated EHV-1 early promoters (EICP0, TK, EICP22, and EICP27) in a dose-dependent manner; (ii) the IR2P abrogated the IEP and the EICP27P (UL5) mediated transactivation of viral promoters in a dose-dependent manner, and (iii) the IR2P, like the IEP itself, also downregulated the IE promoter, indicating that the IEP TAD is not necessary to downregulate the IE promoter. In vitro interaction assays revealed that the IR2P interacts with TATA box-binding protein (TBP). The essential domain(s) of the IR2P that mediate negative regulation were mapped to amino acid residues 1 to 706, indicating that the DNA-binding domain and the NLS of the IR2P may be important for the downregulation. In transient-transfection and virus growth assays, the IR2P reduced EHV-1 production by 23-fold compared to virus titers achieved in cells transfected with the empty vector. Overall, these studies suggest that the IR2P downregulates viral gene expression by acting as a dominant-negative protein that blocks IEP-binding to viral promoters and/or squelching the limited supplies of TFIIB and TBP.