ICP0 translocates USP7 from a nuclear to cytoplasmic protein. (A) 293T cells were transfected with GFP-tagged wt-ICP0 or ICP0.NLS-^mut alone or in combination with DsRed-tagged USP7 at a ratio of at 1:1. Wt-ICP0 was detectable both within the nucleus and cytoplasm (i). On the other hand, wt-USP7 was an entirely nuclear protein (iii). Modification of USP7 by attaching a NES motif allowed it to be expressed both in the nucleus and cytoplasm (iv). In cells coexpressing USP7 and wt-ICP0, USP7 was a predominantly cytoplasmic protein (v) that colocalized with ICP0. As expected, mutation of ICP0 NLS motif excluded it from the nucleus (ii) and abolished its colocalization with USP7 (vi). In these cells, USP7 behaved as in non–ICP0-expressing cells, remaining a nuclear protein. (B) HEK293-TLR4 cells were transfected with DsRed-USP7 and stimulated by LPS. DsRed-tagged USP7 cellular localization was traced over 1 hour after LPS stimulation. USP7 was detectable within the cytoplasm starting 20 minutes after LPS stimulation and lasting for up to an hour. Images were obtained with a Zeiss LSM 510 confocal microscope using 63× water-immersion objective lens.