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A.E. Buckner, C.E. Ekworomadu, R.D. Dix; Nicotine Alters Transcriptional Regulation in Human Cytomegalovirus–Infected Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3052.
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© ARVO (1962-2015); The Authors (2016-present)
Recent animal studies suggest that retinal pigment epithelial (RPE) cells are an ocular site for cytomegalovirus (CMV) latency and reactivation (Zhang et al, IOVS 46:252, 2005). Since tobacco smoking has been identified as a cofactor in a number of chronic diseases, we hypothesized that nicotine may contribute to CMV reactivation from RPE cells, possibly through stimulation of cellular regulatory proteins (Sp1, NF–ΚB, and AP–1) required for transcription of virus and host genes associated with productive, lytic, replication.
Monolayers of ARPE–19 cells were treated with nicotine (100 nM) for 1 hr prior to a 4 hr infection with human CMV (HCMV) [Towne] at an input multiplicity of 1 PFU/cell. Fresh nicotine was added at time of virus infection for a total nicotine exposure time of 5 hrs. Monolayers of untreated and/or uninfected ARPE–19 cells were included as controls. Extracts were prepared from experimental and control cell monolayers and examined by western blot and gel mobility shift assays for detection and semi–quantification of transcription factors Sp–1, NF–ΚB, and AP–1.
Uninfected and HCMV–infected cellular extracts in the absence of nicotine treatment harbored detectable and equivalent amounts of Sp–1, the p65 and p50 subunits of NF–ΚB, and c–Fos and c–Jun of AP–1 as determined by western blot analysis. Significantly, nicotine treatment resulted in a substantial and reproducible increase in protein expression for all three transcription factors in HCMV–infected cells when compared with controls. Gel mobility shift assays of nuclear extracts prepared from nicotine–treated HCMV–infected cells confirmed enhanced protein–DNA binding interactions for Sp1, NF–ΚB, and AP–1 subunits.
Nicotine treatment stimulated Sp1, NF–ΚB, and AP–1 expression within ARPE–19 cells during HCMV infection. The enhanced protein–DNA binding interaction demonstrated that protein expression within nicotine–treated HCMV–inoculated ARPE–19 cells is associated with virus replication rather than mere upregulation of synthesis of transcription factor protein subunits. These findings support the idea that tobacco–acquired nicotine can serve as a cofactor to stimulate HCMV reactivation in RPE cells, thereby contributing to onset of chronic disease. New research is oriented toward determining effects of nicotine on latently infected circulating monocytes.
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