University of Pittsburgh and Children's Hospital of Pittsburgh, One Children's Hospital Drive, 4401 Penn Ave, Pittsburgh, PA 15224, USA.
Virol J. 2009 Dec 29;6:231. doi: 10.1186/1743-422X-6-231.
EBNA3C is an EBV-encoded nuclear protein, essential for proliferation of EBV infected B-lymphocytes. Using EBNA3C amino acids 365-545 in a yeast two hybrid screen, we found an interaction with the Growth Arrest and DNA-damage protein, Gadd34. When both proteins are overexpressed, Gadd34 can interact with EBNA3C in both nuclear and cytoplasmic compartments. Amino acids 483-610 of Gadd34, including the two PP1a interaction, and the HSV-1 ICPgamma34.5 homology domains, are required for the interaction. Furthermore, interaction is lost with a mutant of EBNA3C (509 DVIEVID 515-->AVIAVIA), that abolishes EBNA3C coactivation ability as well as SUMO interaction1. In B-cells, Gadd34, and EBNA3C are present in a complex with PP1a using microcystin sepharose affinity purification, Using a lymphoblastoid cell line in which EBNA3C protein levels are conditional on hydroxytamoxifen, surprisingly, we found that (i) EBNA3C maintains phosphorylation of eIF2alpha at serine 51, and (ii) protects against ER stress induced activation of the unfolded protein response as measured by XBP1 (u) versus XBP1(s) protein expression and N-terminal ATF6 cleavage. In reporter assays, overexpression of Gadd34 enhances EBNA3C's ability to co-activate EBNA2 activation of the LMP1 promoter. Collectively the data suggest that EBNA3C interacts with Gadd34, activating the upstream component of the UPR (eIF2alpha phosphorylation) while preventing downstream UPR events (XBP1 activation and ATF6 cleavage).
EBNA3C 是 EBV 编码的核蛋白，对 EBV 感染的 B 淋巴细胞的增殖至关重要。我们使用酵母双杂交筛选 EBNA3C 的氨基酸 365-545，发现它与生长停滞和 DNA 损伤蛋白 Gadd34 相互作用。当两种蛋白都过表达时，Gadd34 可以在内核和细胞质区室中与 EBNA3C 相互作用。Gadd34 的氨基酸 483-610，包括两个 PP1a 相互作用和 HSV-1 ICPgamma34.5 同源结构域，是相互作用所必需的。此外，与一种突变的 EBNA3C（509DVIEVID515-->AVIAVIA）失去相互作用，该突变会破坏 EBNA3C 的共激活能力以及 SUMO 相互作用 1。在 B 细胞中，Gadd34 和 EBNA3C 与 PP1a 一起存在于一个复合物中，使用微囊藻素琼脂糖亲和纯化。使用一种条件性依赖于羟他莫昔芬的 EBNA3C 蛋白水平的淋巴母细胞系，令人惊讶的是，我们发现：（i）EBNA3C 保持 eIF2alpha 丝氨酸 51 的磷酸化，以及（ii）防止未折叠蛋白反应诱导的 ER 应激激活，如 XBP1（u）与 XBP1（s）蛋白表达和 N 端 ATF6 切割所示。在报告基因实验中，Gadd34 的过表达增强了 EBNA3C 共同激活 EBNA2 激活 LMP1 启动子的能力。总的来说，这些数据表明 EBNA3C 与 Gadd34 相互作用，激活 UPR 的上游成分（eIF2alpha 磷酸化），同时防止 UPR 的下游事件（XBP1 激活和 ATF6 切割）。
