London F S, Brinker J M, Ziaie Z, Kefalides N A
Department of Medicine, University of Pennsylvania, Philadelphia.
Lab Invest. 1990 Feb;62(2):189-95.
Herpes simplex virus type 1 produces an early decrease in protein synthesis in cultured smooth muscle cells isolated from human umbilical artery. To confirm that suppression of host protein synthesis occurs before virus protein expression, cultures were treated with actinomycin D (5 micrograms/ml) continuously from the beginning of infection. In the presence of actinomycin D, by 3.5 hours postinfection, virus-infected cultures showed much less incorporation of [35S]methionine into sodium dodecyl sulfate-polyacrylamide electrophoresis-separated products than did identically treated mock-infected cultures. In the absence of actinomycin D, there was incorporation of radiolabel into viral proteins as early as 4.5 hours postinfection. Hybridization of slot-blotted total RNA demonstrated that virus-infected cultures treated with and without actinomycin D showed a marked decrease in hybridizable RNA by 2 hours PI when compared with mock-infected controls. This decrease was seen with cDNA probes for the mRNA for secreted extracellular matrix macromolecules (fibronectin, thrombospondin and collagen chains alpha 2(I), alpha 1(III] as well as with cDNA probes for RNA of intracellular macromolecules actin and beta-tubulin. In vitro translation of total RNA isolated from infected human smooth muscle cells at 2 and 5 hours postinfection resulted in far less [35S]methionine incorporation into sodium dodecyl sulfate-polyacrylamide electrophoresis-separated products than would be predicted from the amount of hybridizable RNA available. The results suggest that host message is rapidly rendered nonfunctional as well as degraded by means of a virion-competent mechanism(s).
1型单纯疱疹病毒可使从人脐动脉分离出的培养平滑肌细胞中的蛋白质合成在早期减少。为了证实宿主蛋白质合成的抑制发生在病毒蛋白表达之前，从感染开始就用放线菌素D（5微克/毫升）持续处理培养物。在放线菌素D存在的情况下，感染后3.5小时，病毒感染的培养物中[35S]甲硫氨酸掺入十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离产物中的量比同样处理的模拟感染培养物少得多。在没有放线菌素D的情况下，感染后最早在4.5小时就有放射性标记掺入病毒蛋白中。斑点印迹总RNA的杂交显示，与模拟感染对照相比，用和不用放线菌素D处理的病毒感染培养物在感染后2小时可杂交RNA显著减少。用分泌型细胞外基质大分子（纤连蛋白、血小板反应蛋白和胶原链α2（I）、α1（III））的mRNA的cDNA探针以及细胞内大分子肌动蛋白和β-微管蛋白的RNA的cDNA探针都观察到了这种减少。对感染后2小时和5小时从感染的人平滑肌细胞中分离的总RNA进行体外翻译，结果显示[35S]甲硫氨酸掺入十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离产物中的量比根据可用可杂交RNA的量预测的要少得多。结果表明，宿主信息通过一种病毒体相关机制迅速丧失功能并被降解。
