Zhang Y, Sirko D A, McKnight J L
Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pennsylvania 15261.
J Virol. 1991 Feb;65(2):829-41. doi: 10.1128/JVI.65.2.829-841.1991.
The transcriptional induction of the alpha or immediate-early gene class of herpes simplex virus type 1 effected by the alpha trans-induction factor (alpha TIF, ICP25, VP16, Vmw65) requires an alpha-specific cis-acting site. Increased transcription does not result from the direct, independent binding of alpha TIF, but rather from an alpha TIF-dependent formation of a protein-DNA complex containing, in addition to alpha TIF, at least one host cell factor. One of the host factors is a POU domain protein which recognizes an octamer element in the alpha-specific consensus. There is evidence that alpha TIF may drive the formation of multiple protein-DNA complexes containing a POU protein and additional host factors. Previously, the gene products of UL46 and UL47 have been implicated in modulating the alpha TIF-dependent transcriptional induction of alpha genes. Our current studies have extended these analyses from a transient-expression system to a series of viral deletion mutants. In these studies we demonstrate that neither UL46- nor UL47-encoded gene product, either separately or in combination, is required for viral growth in cell culture. The absence of UL47 reduces by up to 80% the ability of the virus to induce an alpha-regulated thymidine kinase reporter gene resident in 143TK- cells. Autoradiograms of [35S]methionine pulse-labeled infected cell proteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, show that deleting UL46 and/or UL47 has no discernable effect on the synthesis of alpha TIF or alpha TIF-containing proteins. Subsequent Western immunoblot analysis, with rabbit anti-alpha TIF antibodies made to an alpha TIF-Staphylococcus aureus protein A fusion, demonstrated that the accumulation and steady-state levels of alpha TIF or alpha TIF-containing proteins was indistinguishable from that of the thymidine kinase-negative isogenic parental virus, R delta 305.
由α反式诱导因子（αTIF、ICP25、VP16、Vmw65）介导的1型单纯疱疹病毒α基因或即刻早期基因类别的转录诱导需要一个α特异性顺式作用位点。转录增加并非源于αTIF的直接、独立结合，而是源于αTIF依赖性的蛋白质-DNA复合物的形成，该复合物除αTIF外还至少包含一种宿主细胞因子。其中一种宿主因子是一种POU结构域蛋白，它识别α特异性共有序列中的八聚体元件。有证据表明，αTIF可能驱动包含POU蛋白和其他宿主因子的多种蛋白质-DNA复合物的形成。此前，UL46和UL47的基因产物已被证明与调节αTIF依赖性的α基因转录诱导有关。我们目前的研究已将这些分析从瞬时表达系统扩展到一系列病毒缺失突变体。在这些研究中，我们证明，无论是单独还是联合，UL46或UL47编码的基因产物都不是病毒在细胞培养中生长所必需的。缺少UL47会使病毒诱导143TK-细胞中α调节的胸苷激酶报告基因的能力降低多达80%。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离的[35S]甲硫氨酸脉冲标记感染细胞蛋白的放射自显影片显示，缺失UL46和/或UL47对αTIF或含αTIF蛋白的合成没有明显影响。随后用针对αTIF-金黄色葡萄球菌蛋白A融合体产生的兔抗αTIF抗体进行的蛋白质免疫印迹分析表明，αTIF或含αTIF蛋白的积累和稳态水平与胸苷激酶阴性的同基因亲本病毒R delta 305没有区别。
