For analysis of UL44 intranuclear localization in HCMVinfected U373-SUMO-1 and U373-Neo cell lines, cells were seeded at 2.5 6 10 5 /well on glass coverslips in 6-well plates and allowed to attach. The next day, cells were infected with HCMV AD169 at an MOI of 1 or of 5 PFU/cell. Cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature, and then permeabilized with acetone for 2 min at -20uC. After washing extensively with PBS, cells were incubated first with 4% FBS in PBS for 1 h at room temperature and then with a primary mouse monoclonal antibody against UL44 (10-C50, Fitzgerald Industries International) at a dilution of 1:100 in FBS 4% in PBS for 1 h at 37uC. Cells were then washed extensively with 4% FBS in PBS and incubated with a secondary goat anti-mouse fluorescein-conjugated antibody (Ig-FITC, Chemicon International) at a dilution of 1:1000 for 1 h at 37uC. Cells were successively washed with PBS and mounted in 70% glycerol in PBS. For better visualization, cells were counterstained with Evans Blue and analyzed also for red fluorescence. CLMS analysis was then performed as described above.