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首页> 外文期刊>American Journal of Physiology >Perinuclear localization of Na-K-Cl-cotransporter protein after human cytomegalovirus infection.
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Perinuclear localization of Na-K-Cl-cotransporter protein after human cytomegalovirus infection.

机译：人巨细胞病毒感染后Na-K-Cl-cotransporter蛋白的核内定位。

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摘要

We (41) previously reported that Na-K-Cl-cotransporter (NKCC) function and microsomal protein expression are both dramatically reduced late in human cytomegalovirus (HCMV) infection of a human fibroblast cell line (MRC-5). We now report DNA microarray data showing that no significant HCMV-dependent NKCC gene repression can be detected 30 h postexposure (PE) to the virus. Consequently, we used plasma membrane biotinylation and subsequent subcellular fractionation in combination with semiquantitative immunoblotting and confocal microscopy to investigate the possibility that intracellular redistribution of the NKCC protein after HCMV infection could be a cause of the HCMV-induced loss of NKCC ion transport function. Our results show that the lifetime of plasmalemmal NKCC protein in quiescent, uninfected MRC-5 cells is approximately 48 h, and <20% of the total expressed NKCC protein are in the plasma membrane. The remainder (approximately 80%) was detected as diffusely distributed, small punctate structures in the cytoplasm. Following HCMV infection: 1) NKCC protein expression in the plasmalemma was sharply reduced (approximately 75%) within 24 h PE and thereafter continued to slowly decrease; 2) total cellular NKCC protein content remained unchanged or slightly increased during the course of the viral infection; and 3) HCMV infection caused NKCC protein to accumulate in the perinuclear region late in the HCMV infection (72 h PE). Thus our results imply that, in the process of productive HCMV infection, NKCC protein continues to be synthesized, but, instead of being delivered to the plasma membrane, it is clustered in a large, detergent-soluble perinuclear structure.
机译：我们（41）以前曾报道，在人类成纤维细胞系（MRC-5）的人类巨细胞病毒（HCMV）感染后期，Na-K-Cl-共转运子（NKCC）功能和微粒体蛋白表达均显着降低。现在，我们报告DNA微阵列数据，显示在暴露于病毒30小时后（PE），没有检测到明显的依赖HCMV的NKCC基因抑制。因此，我们将质膜生物素化和随后的亚细胞分级分离与半定量免疫印迹和共聚焦显微镜相结合，以研究HCMV感染后NKCC蛋白在细胞内的重新分布可能是HCMV诱导的NKCC离子转运功能丧失的原因。我们的结果表明，在静止的，未感染的MRC-5细胞中，质膜NKCC蛋白质的寿命约为48小时，而表达的NKCC蛋白质的总量中<20％在质膜中。其余的（大约80％）被检测为细胞质中弥散分布的小点状结构。 HCMV感染后：1）PE后24小时内血浆浆膜中NKCC蛋白表达急剧降低（约75％），此后继续缓慢降低; 2）在病毒感染过程中，总细胞NKCC蛋白含量保持不变或略有增加; 3）HCMV感染导致NKCC蛋白在HCMV感染后期（PE 72h）积聚在核周区域。因此，我们的结果暗示，在生产性HCMV感染的过程中，NKCC蛋白继续合成，但不是传递到质膜上，而是聚集在大的洗涤剂可溶的核周结构中。

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