Gregory D, Hargett D, Holmes D, Money E, Bachenheimer S L
Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599-7290, USA.
J Virol. 2004 Dec;78(24):13582-90. doi: 10.1128/JVI.78.24.13582-13590.2004.
Infection by herpes simplex virus type 1 (HSV-1) induces a persistent nuclear translocation of NFkappaB. To identify upstream effectors of NFkappaB and their effect on virus replication, we employed mouse embryo fibroblast (MEF)-derived cell lines with deletions of either IKK1 or IKK2, the catalytic subunits of the IkappaB kinase (IKK) complex. Infected MEFs were assayed for virus yield, loss of IkappaBalpha, nuclear translocation of p65, and NFkappaB DNA-binding activity. Absence of either IKK1 or IKK2 resulted in an 86 to 94% loss of virus yield compared to that of normal MEFs, little or no loss of IkappaBalpha, and greatly reduced NFkappaB nuclear translocation. Consistent with reduced virus yield, accumulation of the late proteins VP16 and gC was severely depressed. Infection of normal MEFs, Hep2, or A549 cells with an adenovirus vector expressing a dominant-negative (DN) IkappaBalpha, followed by superinfection with HSV, resulted in a 98% drop in virus yield. These results indicate that the IKK-IkappaB-p65 pathway activates NFkappaB after virus infection. Analysis of NFkappaB activation and virus replication in control and double-stranded RNA-activated protein kinase-null MEFs indicated that this kinase plays no role in the NFkappaB activation pathway. Finally, in cells where NFkappaB was blocked because of DNIkappaB expression, HSV failed to suppress two markers of apoptosis, cell surface Annexin V staining and PARP cleavage. These results support a model in which activation of NFkappaB promotes efficient replication by HSV, at least in part by suppressing a host innate response to virus infection.
单纯疱疹病毒1型（HSV-1）感染可诱导核因子κB（NFκB）持续发生核转位。为了确定NFκB的上游效应分子及其对病毒复制的影响，我们使用了源自小鼠胚胎成纤维细胞（MEF）的细胞系，这些细胞系缺失了IκB激酶（IKK）复合物的催化亚基IKK1或IKK2。对感染的MEF进行病毒产量、IκBα缺失、p65核转位以及NFκB DNA结合活性的检测。与正常MEF相比，缺失IKK1或IKK2均导致病毒产量损失86%至94%，IκBα几乎没有损失或无损失，并且NFκB核转位大幅减少。与病毒产量降低一致，晚期蛋白VP16和gC的积累也严重受抑。用表达显性负性（DN）IκBα的腺病毒载体感染正常MEF、Hep2或A549细胞，随后再用HSV进行超感染，结果病毒产量下降了98%。这些结果表明，IKK-IκB-p65通路在病毒感染后激活NFκB。对对照和双链RNA激活的蛋白激酶缺失的MEF中NFκB激活和病毒复制的分析表明，该激酶在NFκB激活途径中不起作用。最后，在因DN IκB表达而使NFκB被阻断的细胞中，HSV无法抑制凋亡的两个标志物，即细胞表面膜联蛋白V染色和聚（ADP-核糖）聚合酶（PARP）裂解。这些结果支持了一种模型，即NFκB的激活至少部分通过抑制宿主对病毒感染的固有反应来促进HSV的有效复制。
