FIG. 2.

MHV-68 ORF49 encodes a putative homologue of the EBV BRRF1 gene product and is located in both the cytoplasm and the nucleus. (A) Comparison of predicted amino acid sequences of MHV-68 ORF49 with those of homologues in other members of the gammaherpesvirus subfamily. Shown is a comparison of MHV-68 ORF49 (amino acids 30 to 79 and 170 to 219) with a relatively greater homology. (B) Expression of FLAG-tagged ORF49. Vero cell lysates transfected with either vector alone or pFLAG-ORF49 (200 ng) were separated on 12% gel. Monoclonal anti-FLAG antibody was used for Western analysis. The asterisk indicates nonspecific bands used as a loading control. (C, D) Subcellular localization of the ORF49 protein. The Vero cells were transfected with pFLAG-ORF49 (C), pEGFP-C1 (D, upper), or pEGFP-ORF49 (D, lower) and then fixed at 48 h posttransfection. FLAG-tagged ORF49 was analyzed by indirect-immunofluorescence assays with anti-FLAG monoclonal antibody, followed by incubation with rhodamine-conjugated goat anti-mouse IgG (red). The GFP signals (green) from EGFP and EGFP fusion protein were visualized after fixation. (E) Subcellular localization of FLAG-tagged RTA and EGFP-fused ORF49 in cotransfected cells. Vero cells were cotransfected with FLAG-tagged RTA and EGFP-fused ORF49 and subjected to indirect-immunofluorescence assays as described for panels C and D. The nucleus was stained with DAPI (blue). All fluorescence images were obtained with a confocal laser scanning microscope.