To investigate whether sumoylation is indeed involved in the altered intranuclear distribution of UL44, an RNAi approach was employed to suppress the sumoylation system of the cells by silencing Ubc9 since Ubc9 is the only unique and essential enzyme in the SUMO-conjugating pathway [21] . U373-Neo and U373-SUMO-1 cells were transduced with a lentivirus expressing shUbc9, followed by selection with puromycin and induction with doxycyline to establish Ubc9-knocked-down cell lines (U373-Neo shUbc9 and U373-SUMO-1 shUbc9, respectively). As a control, U373-SUMO-1 cells were also transduced with a lentivirus expressing a non-silencing shRNA sequence (U373-SUMO-1 NS). As shown in Figure 6B , Ubc9 was almost completely silenced in cells infected with the shUbc9 lentivirus (U373-SUMO-1 shUbc9) as compared to the cells transduced with the control lentivirus (U373-SUMO-1 NS) and to nontransduced cells (U373-Neo and U373-SUMO-1). To examine the effect of Ubc9 knock-down on intranuclear distribution of UL44, these cell lines were infected with HCMV. As shown in Fig. 6A (upper panels) , upon doxycycline induction, the nuclear UL44 staining in Ubc9-knocked-down U373-SUMO-1 cells was similar to that of U373 cells not overexpressing SUMO-1 (U373-Neo and U373-Neo shUbc9; Fig. 6A , upper panels, and Fig.  S8B ). In contrast, the U373-SUMO-1 cells transduced with the non-silencing lentivirus (U373-SUMO-1 NS, Fig. 6A , upper panels) retained an altered intranuclear distribution of UL44 similar to that observed in the non-transduced cells (U373-SUMO-1) or in the shUbc9-transduced U373-SUMO-1 cells with no doxycycline induction (data not shown). Altogether, these results established that the altered intranuclear distribution of UL44 in HCMV-infected cells upon SUMO-1 overexpression depends on Ubc9-mediated sumoylation.