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A.E. Buckner, R.D. Dix; Human Cytomegalovirus Infection of ARPE-19 Cells Downregulates NF-B Transcriptional Activity . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4618.
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Purpose. Little information is presently available regarding interaction of human cytomegalovirus (HCMV) at the molecular level with various cell types of the retina during AIDS-related CMV retinitis. Previous work has shown that soon after HCMV infection of many non-retinal cell types, transcription factors such as NF-ΚB, AP-1 and Sp1 are induced to regulate viral gene expression. Moreover, NF-ΚB plays an important role in immune and inflammatory responses associated with many viral genes. We therefore explored the effect of HCMV infection on the expression of NF-ΚB in human ARPE-19 cells, a cell line with properties similar to primary cultures of human retinal pigment epithelial (RPE) cells. Methods. ARPE-19 cells were seeded in complete medium at a density of 5 x 105 cells per well in a six-well plate 18-24 hrs prior to transfection. At time of transfection, cells were incubated in serum-free medium, inoculated with HCMV (Towne) at 5 PFU/cell for 1 hr, and NF-ΚB luciferase DNA (pNF-ΚB-Luc) was introduced into the cells by cationic lipid-mediated transfection. Complete medium was added at 3 hrs post-transfection. Cells were harvested at 40-48 hrs post-transfection, and lysed using a reporter lysis buffer. Protein concentration was determined in two separate experiments using the bicinchoninic acid (BCA) protein assay and standardized for each experiment. A luciferase activity assay was performed and data was analyzed using a standard luminometer. Results. ARPE-19 cells transfected with pNF-ΚB-Luc alone exhibited promoter activity at a level of ~30,000 relative light units (RLU). In comparison, cells transfected with pNF-ΚB-Luc in the presence of HCMV infection showed a 15-fold decrease in promoter activity. As expected, a positive control for a mitogen-activated protein kinase (pFC-MEKK) showed a 2-fold increase in luciferase activity. Conclusions. Although NF-ΚB activity was detected in uninfected ARPE-19 cells, a significant decrease was observed in HCMV-infected ARPE-19 cells. This observation supports the finding of Cinatl et al. (2001) that HCMV circumvents NF-ΚB dependence in RPE cells. Whether HCMV causes a similar decrease in NF-ΚB activity in cultures of primary RPE cells remains to be determined.
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