FIG. 6.

RUNX1b precludes E4orf6 localization at the periphery of viral replication centers. The hCAR-transduced mouse A9 cells analyzed in Fig. 5 were infected with the E4orf6/E4orf7 mutant virus dl356. After 24 h, the cells were extracted with Triton X-100 as described in Materials and Methods and processed for double-label immunofluorescence using the E4orf6-specific antibody Rsa#3 and the HA-specific rat antibody. The phenotypically wild-type virus dl356 was used for this experiment, because the Rsa#3 antibody recognizes both E4orf6 protein and the 17-kDa E4orf6/E4orf7 fusion protein. In the absence of staining for the E2A-DBP, viral replication centers can be recognized by differential interference contrast (DIC) illumination or by the absence of DAPI staining in the DNA image. In the merged image, the E4orf6 protein is shown in green and RUNX1 is shown in magenta. DNA was visualized by being stained with DAPI and is shown in blue. Representative micrographs are shown, except for that for the HA-RUNX1a cell in panel d. This cell represents a rare cell displaying (<5%) E4orf6 protein concentrated about the viral replication centers defined by RUNX1 staining. The bar in each DIC image represents 5 μm.