Kim S K, Holden V R, O'Callaghan D J
Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport 71130-3932, USA.
J Virol. 1997 Feb;71(2):1004-12. doi: 10.1128/JVI.71.2.1004-1012.1997.
The equine herpesvirus 1 (EHV-1) immediate-early (IE) phosphoprotein is essential for the activation of transcription from viral early and late promoters and regulates transcription from its own promoter. The EHV-1 EICP22 protein, a homolog of ICP22 of herpes simplex virus, increased the in vitro DNA binding activity of the IE protein for sequences in the IE, early, and late promoters. The EICP22 protein affected the rate as well as the extent of the IE protein binding to promoter DNA sequences. To study the DNA binding activity of the IE protein, Trp493, Gln495, Asn496, and Lys498 of the WLQN region, which is directly involved in DNA binding, were replaced with Ser (IEW493S), Glu (IEQ495E), Ile (IEN496I), and Glu (IEK498E), respectively. Gel shift assays revealed that the glutathione S-transferase (GST)-IEQ495E(407-615) and GST-IEK498E(407-615) proteins failed to bind to the IE promoter, indicating that the Gln and Lys residues are important for the DNA binding activity. In the presence of the GST-EICP22 protein, DNA binding activity of the GST-IEQ495E(407-615) protein was restored, suggesting that the EICP22 protein cooperates with the IE protein to regulate EHV-1 gene expression. Transient-transfection assays also showed that the EICP22 protein allowed the IEQ495E mutant to be functional as a transactivator. These results are unique and may represent an important role for the EICP22 protein in EHV-1 gene regulation.
马疱疹病毒1型（EHV-1）立即早期（IE）磷蛋白对于激活病毒早期和晚期启动子的转录至关重要，并调节其自身启动子的转录。EHV-1 EICP22蛋白是单纯疱疹病毒ICP22的同源物，它增加了IE蛋白对IE、早期和晚期启动子中序列的体外DNA结合活性。EICP22蛋白影响IE蛋白与启动子DNA序列结合的速率和程度。为了研究IE蛋白的DNA结合活性，将直接参与DNA结合的WLQN区域的Trp493、Gln495、Asn496和Lys498分别替换为Ser（IEW493S）、Glu（IEQ495E）、Ile（IEN496I）和Glu（IEK498E）。凝胶迁移试验表明，谷胱甘肽S-转移酶（GST）-IEQ495E（407-615）和GST-IEK498E（407-615）蛋白无法与IE启动子结合，这表明Gln和Lys残基对于DNA结合活性很重要。在存在GST-EICP22蛋白的情况下，GST-IEQ495E（407-615）蛋白的DNA结合活性得以恢复，这表明EICP22蛋白与IE蛋白协同调节EHV-1基因表达。瞬时转染试验还表明，EICP22蛋白使IEQ495E突变体能够作为反式激活因子发挥作用。这些结果是独特的，可能代表了EICP22蛋白在EHV-1基因调控中的重要作用。
