Next, 200 ng of the labeled probe was precipitated along with 6 g Salmon Sperm DNA (Invitrogen) and 4 g Human Cot-1 DNA (Invitrogen) and resuspended in CEP hybridization buffer (55% formamide, 1 SSC, pH 7.0, and 10% dextran sulfate) followed by denaturation at 70 C for 10 min. in vitro activation of peripheral B cells could support, in some cases, detectable but inefficient KSHV contamination (Fig. 1). Contamination of peripheral B cells with KSHV was highest when cells were infected with KSHV before IL-4 and CD40L activation (0.13 0.14% with a maximum of 0.28% of GFP+ cells). In comparison, 0.02 0.02%, 0.03 0.01%, and 0.06 0.06% of B cells were GFP+ when cells were exposed to KSHV, respectively, on days 0, 1, and 3 following B cell activation. These low efficiencies in the presence of IL-4 and CD40L were confirmed as live cells were passaged for 5 doublings and GFP+ cells could still be detected by flow cytometry. While an earlier study (32) reported up to 30% of activated peripheral B cells to be susceptible to contamination, although we IOX 2 infected similarly activated, peripheral B cells from multiple donors in 10 individual experiments, we found no more than 0.13% of them to be infected with KSHV (Fig. 1). EBV Contamination Supports an Optimal Contamination of Peripheral B Cells by KSHV. We tested whether EBV coinfection supports an optimal contamination of peripheral B cells with KSHV. Peripheral CD19+ B cells were infected with the B95-8 strain of EBV at an MOI of 1 1 to 2 2. An average of 70% of the cells were blasts at day 3 or 4 4 postinfection with EBV (common from 7 impartial biological replicates). CD19+ B cells were exposed to KSHV BAC16 at different time points before, on the same day as, or following EBV contamination. EBV infection promoted optimal contamination (up to a 20-fold increase relative to activated B cells) of peripheral B cells with KSHV (up to 2.50 1.13%) (Fig. 2 and and and 0.05; ** 0.01 by Wilcoxon rank sum test. We examined what EBV contributes to foster KSHV contamination. First, does EBV induce a potential KSHV entry receptor? DC-SIGN, also called CD209, was reported to be a receptor for KSHV entry in peripheral CD19+ B cells (32) and Rabbit polyclonal to TGFbeta1 is the only potential receptor for KSHV previously described to be on B lymphocytes (33, 34). We used 2 different antiCDC-SIGN antibodies and validated them around the human monocytic THP-1 cell line (35, 36) ( 0.05 by SenCAdichie test. (and and were IOX 2 repeatedly sorted for GFP+ cells (KSHV+/EBV+-fast and KSHV+/EBV+-slow cells, respectively) (gray arrows) and harvested in TRIzol at different time points. Following isolation of DNA, the average number of EBV and KSHV genomes per cell was quantified by qPCR. Cells from unsorted and sorted populations were collected at different time points and analyzed for IOX 2 the presence of KSHV and EBV by qPCR (Fig. 4and and 0.05 and fold-change ?1.5 or 1.5) between the KSHV+/EBV+-fast and KSHV+/EBV+-slow cells for each sample. Genes shown in heatmaps were ordered by value (the gene with the smallest value is around the left of the heatmap). ( 0.001) (Fig. 7 0.001, false-discovery rate q-value 0.001. ( 0.001, false-discovery rate q-value 0.001, normalized enrichment score = 3.8. Using GSEA, we have also identified cellular genes expressed at higher levels in the KSHV+/EBV+-fast relative to the KSHV+/EBV+-slow cells and found that genes in the pathway involving NF-B were up-regulated in the KSHV+/EBV+-fast cells ( 0.001) (Fig. 7 3 was tested in 3 to 4 4 different donors. Peripheral CD19+ B cells were purified from PBMCs by unfavorable selection according to the instructions of the manufacturer (B Cell Isolation Kit II, Human, Miltenyi Biotec). The isolation yielded 90% CD19+ B cells as determined by staining with anti-CD19 monoclonal antibody and flow cytometry. Cell Culture. The 293 (58) and Daudi (59) cell lines were used for titration of KSHV and the.