FIG 2.

EBV type 2 infects T cells. T cells were purified from PBMCs of 3 or 4 independent donors. (A) T cells were infected with EBV-2 isolated from the Jijoye or BL5 cell line or were cultured in medium only. (B) T cells were infected with EBV-2 (Jijoye derived) in the absence of drugs or in the presence of acyclovir or PAA. (A and B) T cells were harvested 1, 4, 7, and 12 days postinfection for DNA extraction, and multiplex qPCR for the EBV genome and actin was performed to determine the EBV copy number per microgram of DNA (n = 3 for each time point). (C) Real-time limiting-dilution PCR for the viral genome was utilized to determine the frequency of T cells that harbored EBV-2. Primers and probes specific for EBV-2 EBNA-3c were used to detect the EBV-2 genome. The frequency of cells positive for the viral genome was calculated by Poisson distribution analysis of mean data (n = 3). Samples containing 10, 1, 0.1, or no copies of EBNA3c DNA were included as controls. The line at 63.2% indicates the point at which one viral genome-positive cell per reaction was predicted to occur. The x axis shows the numbers of cells per reaction; the y axis shows the percentages of 12 reactions positive for the viral genome. The error bars represent means ± standard errors. (D) Immunofluorescent staining of T cells was performed at 7 days p.i. Mock- and Jijoye-infected T cells were stained to visualize CD3 (green) and LMP-1 (red) on the cell surface and with DAPI to visualize nuclei (blue). The images were captured with a 100× objective using a Nikon Eclipse Ti inverted microscope. (E) Whole-cell lysates from EBV-2- and mock-infected T cells were prepared at 1, 4, 7, and 12 days postinfection (dpi) and subjected to immunoblotting to detect LMP-1 (42 kDa), LMP-2 (50 kDa), EBNA-1 (70 kDa), EBNA-2 (75 kDa), and actin (42 kDa). The data shown are for noninfected (media) and EBV-2 (Jijoye)-infected samples from 1 of 4 individual donors for each time point. As shown, an EBV-2 LCL was used as a positive-staining control.