The equine herpesvirus-1 IR3 gene that lies antisense to the sole immediate-early (IE) gene is trans-activated by the IE protein, and is poorly expressed to a protein
The equine herpesvirus-1 IR3 gene that lies antisense to the sole immediate-early (IE) gene is trans-activated by the IE protein, and is poorly expressed to a protein
复制标题
DOI：
10.1016/j.virol.2007.01.024
复制
发表时间：
2007-06-20
期刊：
影响因子：
3.7
通讯作者：
O'Callaghan, Dennis J.
中科院分区：
文献类型：
--
作者：
Ahn, Byung Chul;Breitenbach, Jonathan E.;O'Callaghan, Dennis J.
马疱疹病毒 1 (EHV-1) 的独特 IR3 基因表达为晚期 1.0-kb 转录本。先前的研究证实了 JR3 转录起始位点，并初步鉴定了 JR3 特有的其他顺式作用元件，如 TATA 盒、443 碱基对 5' 非翻译区 (UTR)、285 碱基对开放阅读框 (ORF) 和 a.poly 腺苷酸化 (A) 信号 [Holden, V.R.、Harty, R.N.、Yalamanchili, R.R., O'Callaghan, D.J., 1992。1 型马疱疹病毒的 IR3 基因：受立即早期基因内含子内序列调控的独特基因。 DNA 序列。 3、143-152]。瞬时转染测定表明，JR3 启动子被 IE 蛋白 (IEP) 强烈反式激活，并且 IEP 与早期 EICP0 和 IR4 调节蛋白的共表达导致 IR3 启动子的最大反式激活。凝胶迁移分析表明 IEP 直接结合 IR3 启动子区域。 Western blot分析表明，IR3合成肽的抗体检测到了大肠杆菌中产生的IR3蛋白；然而，即使通过RNA印迹检测到了IR3转录物，这些上述抗IR3抗体在EHV-I感染的细胞提取物中也没有检测到IR3蛋白。这些发现表明 IR3 可能不表达为蛋白质。未观察到IR3/GFP融合基因的表达，但通过荧光显微镜检测到GFP/IR3融合基因的表达。在进一步尝试使用抗GFP抗体检测IR3/GFP融合蛋白时，蛋白质印迹分析显示在体内未检测到IR3/GFP融合蛋白。有趣的是，从 GFP/IR3 融合基因合成了截短形式的 GFP/IR3 蛋白。然而，预测大小的 GFP/IR3 和 IR3/GFP 融合蛋白是通过融合基因的体外偶联转录和翻译合成的，表明 IR3 蛋白在体内的表达较差。讨论了 IR3 转录本在 EHV-I 感染中的可能作用。 (C) 2007 Elsevier Inc. 保留所有权利。
The unique IR3 gene of equine herpesvirus 1 (EHV-1) is expressed as a late 1.0-kb transcript. Previous studies confirmed the JR3 transcription initiation site and tentatively identified other cis-acting elements specific to JR3 such as a TATA box, a 443 base pair 5'untranslated region (UTR), a 285 base pair open reading frame (ORF), and a.poly adenylation (A) signal [Holden, V.R., Harty, R.N., Yalamanchili, R.R., O'Callaghan, D.J., 1992. The IR3 gene of equine herpesvirus type 1: a unique gene regulated by sequences within the intron of the immediate-early gene. DNA Seq. 3, 143-152]. Transient transfection assays revealed that the JR3 promoter is strongly trans-activated by the IE protein (IEP) and that coexpression of the IEP with the early EICP0 and IR4 regulatory proteins results in maximal trans-activation of the IR3 promoter. Gel shift assays revealed that the IEP directly binds to the IR3 promoter region. Western blot analysis showed that the IR3 protein produced in E. coli was detected by antibodies to IR3 synthetic peptides; however, the IR3 protein was not detected in EHV- I infected cell extracts by these sai ne anti-IR3 antibodies, even though the IR3 transcript was detected by northern blot. These findings suggest that the IR3 may not be expressed to a protein. Expression of an IR3/GFP fusion gene was not observed, but expression of a GFP/IR3 fusion gene was detected by fluorescent microscopy. In further attempts to detect the IR3/GFP fusion protein using anti-GFP antibody, western blot analysis showed that the IR3/GFP fusion protein was not detected in vivo. Interestingly, a truncated form of the GFP/IR3 protein was synthesized from the GFP/IR3 fusion gene. However, GFP/IR3 and IR3/GFP fusion proteins of the predicted sizes were synthesized by in vitro coupled transcription and translation of the fusion genes, suggesting poor expression of the IR3 protein in vivo. The possible role of the IR3 transcript in EHV- I infection is discussed. (C) 2007 Elsevier Inc. All rights reserved.