FIG. 5.
Aggregation of pp65-GFP in transfected COS7 cells is prevented by UL97 kinase activity. Transient expression of a pp65 GFP fusion protein in COS7 cells results in the formation of large nuclear aggregates (A) 24 h posttransfection. Resulting aggregates fluoresce so brightly that they are detected with the filter used for DAPI staining (B). Transiently expressed ppUL97 with a V5 epitope tag localizes to the nucleus (C), whereas the same ppUL97-V5 fusion protein with the K355M point mutation localizes both to the nucleus and to the cytoplasm (D) as shown in the images merged with DAPI staining. Coexpression of pp65-GFP (E) and ppUL97-V5 (F) eliminates the formation of nuclear pp65-GFP aggregates and instead results in the diffuse nuclear localization of both proteins as shown in the merged image (H) with DAPI (G). The inhibition of pp65 aggregation by ppUL97 is antagonized by the addition of MBV, and pp65-GFP is formed (I to L). The inhibition of UL97 kinase activity by MBV allows both the formation of pp65-GFP aggregates (I) and the recruitment of ppUL97 to the aggregates (J) as shown in the merged image (L) with DAPI (K). Similarly, the coexpression of ppUL97-V5-K355M (N) is unable to stop the aggregation of pp65-GFP (M), and the kinase-deficient ppUL97 fusion protein is recruited to the nuclear inclusions as shown in the merged image (P) with DAPI (O).