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J. PML isoforms, we showed that individual PML isoforms were sufficient to suppress EBV lytic reactivation, although PML isoform IV (PML IV) was ineffective because it was most efficiently degraded by EBNA1. Our results provide the first function for EBNA1 in lytic contamination and show that EBNA1 interactions with PML IV lead to a loss of PML nuclear body (NBs) that promotes lytic contamination. INTRODUCTION Epstein-Barr computer virus (EBV) is usually a gammaherpesvirus that infects most people worldwide and is maintained for life through a combination of latent and FIIN-2 lytic modes of contamination in B lymphocytes and epithelial cells. While EBV is usually most often found in a latent mode of contamination in B cells, lifelong persistence of EBV in infected individuals involves occasional reactivation of the virus to the lytic state, and a partial (or abortive) lytic contamination also appears to be important in EBV-induced B-cell and epithelial tumors (8, 31, 32, 66). In addition, in epithelial cells of the orthopharynx, reactivation of EBV to lytic replication is necessary to produce the viral particles responsible for host-to-host spread (50). However, it is currently unclear how viral reactivation is usually brought on value 0.01. (B) Equivalent amounts of cell lysates from untreated AGS-EBV (lane 3) or AGS-EBV treated with siGFP, siEBNA1, siEBNA1a, or AllStars siRNA (siAS) were compared by Western blotting using antibodies for EBNA1, BMRF1, BZLF1, and actin (loading control). (C) Steady-state transcript levels of Rabbit Polyclonal to DDX3Y BZLF1 were monitored by isolating total RNA, synthesizing cDNA, and quantifying transcript levels using primers specific to BZLF1. Values were normalized to the GAPDH housekeeping gene. Average levels of the BZLF1 transcript from three individual experiments are shown for siEBNA1 treatment (with standard deviations) relative to those for the FIIN-2 siGFP treatment (set to 1 1). (D) EBV genomic DNA was quantified by isolating total DNA, amplifying the EBV DS sequence, and normalizing to GAPDH. Average levels (with standard deviations) for siEBNA1 treatment were compared to those for siGFP treatment (set to 1 1; left graph), and results for siEBNA1a and siAllStars treatment was compared to results for untreated AGS-EBV (set to 1 1; left panel). ***, 0.001. Immunofluorescence microscopy. Cells produced on coverslips were fixed using 3% formaldehyde for 15 to 20 min at room temperature and then rinsed briefly using phosphate-buffered saline (PBS). Cells were permeabilized using 1% Triton in PBS for 5 min, followed by two 5-min rinses in PBS. Coverslips were FIIN-2 blocked for 20 min in 4% bovine serum albumin (BSA) in PBS. Samples were incubated with main antibodies against EBNA1 (R4 rabbit serum at 1:300 [30]), PML (PG-M3 at 1:50; Santa Cruz), or BZLF1 (1:100 of monoclonal antibody [Jaap Middeldorp] or 1:50 monoclonal antibody sc-53904 [Santa Cruz]) for 30 min in the humidifying chamber and rinsed twice for 5 min (each) in PBS. Samples were incubated with the secondary antibodies goat anti-rabbit Alexa Fluor 555 (1:800; Molecular Probes) and goat anti-mouse Alexa Fluor 488 (1:800; Molecular Probes) in 4% BSA for 30 min and washed twice for 5 min (each). Coverslips were mounted on slides using ProLong Platinum antifade medium made up of 4,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Images were obtained using the 40 oil objective on a Leica inverted fluorescence microscope and processed using the OpenLAB (ver.X.0) software program. BZLF1-positive cells were quantified by counting more than 100 cells per sample, and experiments were performed in triplicate. Western blotting. Cells were lysed in either 9 M ureaC10 mM Tris (pH 6.8) followed by sonication or in RIPA (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM MgCl2, 10% glycerol, 1% Triton FIIN-2 X-100, and protease inhibitors) for 20 min on ice. Thirty to fifty micrograms of clarified lysates were loaded onto a 10% SDS-PAGE gel and transferred onto nitrocellulose. Membranes were blocked in 5% nonfat dry milk in PBT-T (PBS with 0.1% Tween) for 1 h, followed by incubation with primary antibody in blocking buffer overnight at room heat. The primary antibodies used were EBNA1 K67-3 (1:1000; a.