To investigate whether the nature of DNA could influence the stimulation of UL44 sumoylation, we also performed in vitro sumoylation reactions in the presence of different DNA substrates. Similar stimulation levels were obtained when different dsDNAs were added, regardless of their sequence (data not shown), in keeping with previous observations that UL44 binds DNA in a sequence-independent manner [7] . In contrast, consistently less stimulation of UL44 sumoylation was observed in the presence of ssDNA (Fig. 4A, right panel) , for which UL44 has been shown to possess an apparent affinity lower than for dsDNA [7] . This suggested that UL44 sumoylation could depend on binding to To analyze UL44 sumoylation in vitro, purified 6His-UL44 was incubated in the absence or the presence of sumoylation enzymes and either wild-type SUMO-1 (SUMO-1 wt) or a mutant form of SUMO-1 (SUMO-1 mut) which cannot be covalently linked to substrates. The reaction products were analyzed by western blotting with anti-UL44 and anti-SUMO-1 antibodies. As a positive control, in vitro sumoylation of p53 was also analyzed. (B) Purified 6His-UL44 was incubated in the absence or the presence of sumoylation enzymes and either SUMO-2 or SUMO-3 and analyzed by western blotting with anti-UL44 and anti-SUMO-2/23 antibodies. For all panels, the arrowhead indicates the unmodified form of UL44 or p53 and the asterisks indicate the respective sumoylated forms. doi:10.1371/journal.pone.0049630.g002 DNA. To test this hypothesis, we analyzed the ability of two UL44 mutants, FLAG-UL44Dloop and FLAG-UL44L86A/L87A, which are defective for DNA binding [36] , to be modified by SUMO-1 in mammalian cells. FLAG-UL44Dloop contains three point mutations within a UL44 flexible loop ( 163 HTRVKRNVKKAP 174 ) involved in UL44-DNA interaction [8, 9] , and is therefore impaired in DNA binding [36] . FLAG-UL44L86A/L87A carries two point mutations preventing dimerization of UL44 and strongly impairing the UL44-DNA interaction both in vitro and in vivo [9, 36, 60] . Both FLAG-UL44Dloop (Fig. 4B) and FLAG-UL44L86A/L87A (Fig. 4C) , when coexpressed with HA-SUMO-1 and Ubc9, exhibited strongly reduced sumoylation levels when compared to FLAG-UL44. Importantly, co-immunoprecipitation experiments demonstrated that the reduced sumoylation of FLAG-UL44Dloop and FLAG-UL44L86A/L87A was not due to an impairment of binding of the mutant proteins to Ubc9, since the two mutants precipitated with endogenous Ubc9 at levels comparable to those of the wild-type protein (Fig. 4D ). In addition, the presence of DNA did not stimulate the sumoylation of the UL44Dloop or UL44L86A/L87A mutants in in vitro reactions (Fig. 4E) . Finally, since the UL44Dloop mutant contains a substitution (K167N) involving a potential SUMO target lysine, we wished to exclude the possibility that the reduced sumoylation levels of FLAG-UL44Dloop might be due to alteration of a putative SUMO acceptor site rather than an impairment of DNA binding. To this end, the K167 residue was conservatively mutated to arginine and the mutant protein was tested for in vitro sumoylation in the presence of DNA. The K167R mutant showed a SUMO-1 modification pattern identical to that of wild-type UL44 (Fig. 4F) .