Prod'hon C, Machuca I, Berthomme H, Epstein A, Jacquemont B
Laboratoire de Neurovirologie Moléculaire, Université Claude Bernard, Lyon, France.
Virology. 1996 Dec 15;226(2):393-402. doi: 10.1006/viro.1996.0667.
Previous work has shown that the 68-kDa immediate-early protein of herpes simplex virus type 1 (HSV-1), also known as ICP22, is involved in the control of viral gene expression, although the precise mechanism remains to be elucidated. In order to study the function(s) of this protein, we constructed expression vectors containing the coding sequence of the ICP22 gene placed under the control of the SV40 or HCMV promoter. After cell transfection, ICP22 synthesis was studied by immunoblotting, using a specific antiserum. In transient expression experiments in COS cells in which the ICP22 vector was under the control of the SV40 promoter, we found that ICP22 was able to inhibit chloramphenicol acetyltransferase (CAT) expression under the control of either the alpha 22 (IE4) promoter or other immediate-early promoters, such as alpha 4 (IE3), alpha 0 (IE1), and alpha 27 (IE2). CAT expression under the control of the alpha 4 (IE3) promoter was inhibited in these cells by expression of ICP22 under the control of the HCMV promoter; it was also inhibited in RAT-1 cells by ICP22 expressed under the control of the SV40 or HCMV promoter. In contrast, CAT expression directed by the SV40 or HCMV promoters was only weakly or not inhibited by the ICP22 vectors. We also constructed an expression vector for UL13, a gene whose product is implicated in the phosphorylation of ICP22. Although CAT expression under the control of the alpha 4 (IE3) promoter was also negatively regulated by the UL13 gene product, the effects of the ICP22 (directed by the SV40 or HCMV promoter) and UL13 vectors were not synergistic; furthermore, at a particular molar ratio of the two vectors, inhibition of CAT activity was partially reversed. The results in the present work suggest that ICP22 can negatively regulate the expression of immediate-early viral genes and that its phosphorylation by UL13 protein kinase might be involved in the modulation of its function.
先前的研究表明，单纯疱疹病毒1型（HSV-1）的68-kDa即刻早期蛋白，也称为ICP22，参与病毒基因表达的调控，尽管其确切机制仍有待阐明。为了研究该蛋白的功能，我们构建了表达载体，其中ICP22基因的编码序列置于SV40或HCMV启动子的控制之下。细胞转染后，使用特异性抗血清通过免疫印迹法研究ICP22的合成。在ICP22载体受SV40启动子控制的COS细胞瞬时表达实验中，我们发现ICP22能够抑制α22（IE4）启动子或其他即刻早期启动子（如α4（IE3）、α0（IE1）和α27（IE2））控制下的氯霉素乙酰转移酶（CAT）表达。在这些细胞中，HCMV启动子控制下的ICP22表达抑制了α4（IE3）启动子控制下的CAT表达；在RAT-1细胞中，SV40或HCMV启动子控制下表达的ICP22也抑制了CAT表达。相比之下，SV40或HCMV启动子指导的CAT表达仅受到ICP22载体的微弱抑制或未受抑制。我们还构建了UL13的表达载体，该基因的产物与ICP22的磷酸化有关。尽管α4（IE3）启动子控制下的CAT表达也受到UL13基因产物的负调控，但ICP22（由SV40或HCMV启动子指导）和UL13载体的作用并非协同；此外，在两种载体的特定摩尔比下，CAT活性的抑制部分被逆转。目前的研究结果表明，ICP22可以负向调节病毒即刻早期基因的表达，并且其被UL13蛋白激酶磷酸化可能参与其功能的调节。
