Krajcsi P, Dimitrov T, Hermiston T W, Tollefson A E, Ranheim T S, Vande Pol S B, Stephenson A H, Wold W S
Department of Molecular Microbiology and Immunology, St. Louis University Schoolof Medicine, Missouri 63104, USA.
J Virol. 1996 Aug;70(8):4904-13. doi: 10.1128/JVI.70.8.4904-4913.1996.
Tumor necrosis factor (TNF) is an inflammatory cytokine that inhibits the replication of many viruses in cultured cells. We have reported that adenovirus (Ad) infection of TNF-resistant mouse cells renders them susceptible to lysis by TNF and that two sets of proteins encoded by the E3 transcription unit block TNF cytolysis. The E3 protein sets are named E3-14.7K (14,700 kDa) and E3-10.4K/14.5K (a complex of two proteins of 10,400 and 14,500 kDa). TNF activation of the 85-kDa cytosolic phospholipase A2 (cPLA2) is thought to be essential for TNF cytolysis (i.e.,TNF-induced apoptosis). Here we provide evidence that cPLA2 is important in the response of Ad-infected cells to TNF and that the mechanism by which E3-14.7K and E3-10.4K/14.5K inhibit TNF cytolysis is by inhibiting TNF activation of cPLA2. cPLA2 cleaves arachidonic acid (AA) specifically from membrane phospholipids; therefore, cPLA2 activity was measured by the release of 3H-AA from cells prelabeled with 3H-AA. Uninfected cells or cells infected with wild-type Ad were not lysed and did not release 3H-AA in response to TNF. In contrast, TNF treatment induced cytolysis and 3H-AA release in uninfected cells sensitized to TNF by treatment with cycloheximide and also in infected cells sensitized to TNF by expression of E1A. In C127 cells, in which either E3-14.7K or E3-10.4K/14.5K inhibits TNF cytolysis, either set of proteins inhibited TNF-induced release of 3H-AA. In C3HA cells, in which E3-14.7K but not E3-10.4K/14.5K prevents TNF cytolysis, E3-14.7K but not E3-10.4K/14.5K prevented TNF-induced release of 3H-AA. When five virus mutants with lesions in E3-14.7K were examined, there was a perfect correlation between a mutant's ability to inhibit both TNF-induced cytolysis and release of 3H-AA. E3-14.7K expressed in two stably transfected C127 cell lines prevented both TNF-cycloheximide-induced cytolysis and release of 3H-AA. The E3 proteins also prevented TNF-induced cytolysis and release of 3H-AA in mouse L929 cells, which are spontaneously sensitive to TNF. TNF cytolysis was blocked by dexamethasone, an inhibitor of PLA2 activity, and by nordihydroquaiaretic acid, which inhibits the metabolism of AA to the leukotrienes. Indomethacin, which blocks the formation of prostaglandins from AA, did not inhibit TNF cytolysis. The leukotrienes and prostaglandins are amplifiers of the inflammatory response. We propose that E3-14.7K and E3-10.4K/14.5K function independently in Ad infection to inhibit both cytolysis and inflammation induced by TNF.
肿瘤坏死因子（TNF）是一种炎症细胞因子，可抑制培养细胞中多种病毒的复制。我们曾报道，对TNF具有抗性的小鼠细胞感染腺病毒（Ad）后，会使其易被TNF裂解，且E3转录单元编码的两组蛋白质可阻断TNF的细胞溶解作用。这两组E3蛋白分别被命名为E3 - 14.7K（14,700 kDa）和E3 - 10.4K/14.5K（一种由10,400 kDa和14,500 kDa两种蛋白质组成的复合物）。85 kDa的胞质磷脂酶A2（cPLA2）的TNF激活被认为是TNF细胞溶解（即TNF诱导的细胞凋亡）所必需的。在此，我们提供证据表明，cPLA2在Ad感染细胞对TNF的反应中起重要作用，且E3 - 14.7K和E3 - 10.4K/14.5K抑制TNF细胞溶解的机制是通过抑制cPLA2的TNF激活。cPLA2可特异性地从膜磷脂中裂解花生四烯酸（AA）；因此，通过测量用3H - AA预标记细胞中3H - AA的释放来检测cPLA2活性。未感染的细胞或感染野生型Ad的细胞不会被裂解，且对TNF无反应而释放3H - AA。相反，TNF处理会诱导经放线菌酮处理而对TNF敏感的未感染细胞以及通过表达E1A而对TNF敏感的感染细胞发生细胞溶解并释放3H - AA。在C127细胞中，E3 - 14.7K或E3 - 10.4K/14.5K均可抑制TNF细胞溶解，这两组蛋白质中的任何一组均可抑制TNF诱导的3H - AA释放。在C3HA细胞中，E3 - 14.7K可阻止TNF细胞溶解，而E3 - 10.4K/14.5K则不能，E3 - 14.7K可阻止TNF诱导的3H - AA释放，而E3 - 10.4K/14.5K则不能。当检测五个在E3 - 14.7K中有损伤的病毒突变体时，突变体抑制TNF诱导的细胞溶解和3H - AA释放的能力之间存在完美的相关性。在两个稳定转染的C127细胞系中表达的E3 - 14.7K可同时阻止TNF - 放线菌酮诱导的细胞溶解和3H - AA释放。E3蛋白还可阻止TNF诱导的小鼠L929细胞（对TNF自发敏感）的细胞溶解和3H - AA释放。TNF细胞溶解被PLA2活性抑制剂地塞米松以及抑制AA代谢为白三烯的去甲二氢愈创木酸所阻断。阻断AA形成前列腺素的吲哚美辛不抑制TNF细胞溶解。白三烯和前列腺素是炎症反应的放大剂。我们提出，E3 - 14.7K和E3 - 10.4K/14.5K在Ad感染中独立发挥作用，以抑制TNF诱导的细胞溶解和炎症。
