FIG. 2.

Inhibition of pp71 and GFP-GCP170* aggregate formation by UL97 kinase. Aggregating proteins pp65-GFP, pp71-V5, and GFP-GCP170* were coexpressed in COS7 cells with epitope-tagged pUL97 and the kinase-negative K355M point mutant. Aggregates formed by GFP fusion proteins pp65-GFP (A) and GFP-GCP170* (G) were visualized directly, while pp71-V5 aggregates (D) were stained with a monoclonal antibody to the epitope tag and a FITC-conjugated secondary antibody (green staining). The expressions of epitope-tagged pUL97 and the K355M point mutant were detected with the V5 monoclonal antibody labeled with the Zenon Texas Red labeling kit (red staining). Aggregate formation by pp65-GFP was inhibited in cells that also expressed pUL97 (compare panels A and B). (C) The K355M kinase-negative form pUL97 was unable to inhibit the aggregation of pp65-GFP and was recruited to the aggregates as shown by the yellow staining in the merge panel. The aggregation of pp71 was also inhibited by pUL97 (compare panels D and E). (F) This effect was kinase dependent, since the K355M mutant was unable to reduce pp71 aggregate formation and instead was recruited to these structures, as shown in the merge panel. Aggresomes induced through the expression of a cellular marker for these structures, GFP-GCP170*, were also substantially reduced by the expression of pUL97 (compare panels G and H). (I) The inhibition of aggresome formation by pUL97 appeared to be kinase dependent, since their frequency and distribution were unaffected by the K355M mutant. Interestingly, the K355M mutant was specifically recruited to cytoplasmic aggresomes and was not recruited to nuclear aggresomes, as shown in the merge panel.