FIGURE 6.

EBV establishes latency in B-cells via an abortive lytic infection in the pre-latent phase. (A) Schematic representation of the genetic elements in the Flpe-FRT system. Flpe recombinase can recombine FRT sites in the ubiquitously expressed reporter construct to remove the STOP (neomycin resistant gene and polyA signal; neo-pA) cassette. Upon removal of this STOP, the reporter DsRed is expressed in the cells and all their progeny. (B) DsRed was expressed in the presence of Flpe. Akata/FNF-DsRed reporter cells were transfected with a Flpe expression plasmid. Scale bar, 100 μm. (C) Schematic diagram of recombinant EBV (EBV/BMRF1p-Flpe) construction. The Neo/St cassette, containing neomycin resistance and streptomycin sensitivity genes, was inserted between nucleotides 1 and 505 of the BMRF1 gene to prepare an intermediate, and this was replaced with the Flpe sequence to construct the EBV/BMRF1p-Flpe, which expresses Flpe under the control of the BMRF1 promoter (left). Successful recombination was confirmed by the electrophoresis of the EBV-BAC after BamHI and EcoRI digestion (right). (D) Continuous tracing of abortive lytic cells with recombinant EBV. Akata/FNF-DsRed cells were infected with EBV/BMRF1p-Flpe. Two days after infection, hygromycin was added to the medium to select infected cells. Infected cells were continuously observed (arrowheads) until 17 dpi. The number of DsRed-positive and EGFP-positive cells was counted at indicated time points. Results shown are the means ± SD of three independent experiments. Images were obtained at 17 dpi. Asterisk (*) indicates p < 0.05; dpi, days post-infection. Scale bar, 50 μm.