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.