Pizer L I, Tedder D G, Betz J L, Wilcox K W, Beard P
J Virol. 1986 Dec;60(3):950-9. doi: 10.1128/JVI.60.3.950-959.1986.
In vitro transcription assays were carried out by using as templates DNAs cut from the herpes simplex virus early glycoprotein D gene, the late glycoprotein C gene, the late VP5 gene, and the immediate-early ICP22 gene. Nuclear extracts from suspension cultures of uninfected HeLa cells effectively synthesized RNAs from genes of the immediate-early and delayed-early classes. To a lesser extent, the extracts also used DNAs cut from the late genes as templates. Transcription from the immediate-early gene was inhibited in extracts prepared from infected cells. Analysis of the proteins in infected-cell extracts by gel electrophoresis, transfer to nitrocellulose, and probing with specific antibody demonstrated the presence of the viral regulatory protein ICP4. Chromatographic fractionation of nuclear extract from infected cells yielded a mixture of proteins (fraction VIII) enriched in ICP4 (S.W. Faber and K.W. Wilcox, Nucleic Acids Res., 14:6067-6083, 1986). Addition of fraction VIII to the in vitro assay affected transcription. Depending on the DNA in the assay, an inhibitory or stimulatory effect was observed. Inhibition of RNA synthesis was found when DNA from the immediate-early gene was used as a template, and stimulation was found when DNA from the early or late gene was used.
体外转录试验是以从单纯疱疹病毒早期糖蛋白D基因、晚期糖蛋白C基因、晚期VP5基因和立即早期ICP22基因切割得到的DNA为模板进行的。来自未感染的HeLa细胞悬浮培养物的核提取物能有效地从立即早期和延迟早期类别的基因合成RNA。提取物也能在较小程度上以从晚期基因切割得到的DNA为模板。在从感染细胞制备的提取物中，立即早期基因的转录受到抑制。通过凝胶电泳分析感染细胞提取物中的蛋白质，将其转移至硝酸纤维素膜上，并用特异性抗体进行检测，结果表明存在病毒调节蛋白ICP4。对感染细胞核提取物进行色谱分级分离，得到了富含ICP4的蛋白质混合物（第八组分）（S.W. 费伯和K.W. 威尔科克斯，《核酸研究》，14:6067 - 6083，1986）。将第八组分添加到体外试验中会影响转录。根据试验中所用的DNA，会观察到抑制或刺激作用。当以立即早期基因的DNA为模板时，发现RNA合成受到抑制；当以早期或晚期基因的DNA为模板时，发现有刺激作用。
