The Mre11 Cellular Protein Is Modified by Conjugation of Both SUMO-1 and SUMO-2/3 during Adenovirus Infection

The adenovirus type 5 (Ad5) E1B 55？kDa and E4 Orf6 proteins assemble a Cullin 5-E3 ubiquitin (Ub) ligase that targets, among other cellular proteins, p53 and the Mre11-Rad50-Nbs1 (MRN) complex for degradation. The latter is also inhibited by the E4 Orf3 protein, which promotes the recruitment of Mre11 into specific nuclear sites to promote viral DNA replication. The activities associated with the E1B 55？kDa and E4 Orf6 viral proteins depend mostly on the assembly of this E3-Ub ligase. However, E1B 55？kDa can also function as an E3-SUMO ligase, suggesting not only that regulation of cellular proteins by these viral early proteins may depend on polyubiquitination and proteasomal degradation but also that SUMOylation of target proteins may play a key role in their activities. Since Mre11 is a target of both the E1B/E4 Orf6 complex and E4 Orf3, we decided to determine whether Mre11 displayed similar properties to those of other cellular targets, in Ad5-infected cells. We have found that during Ad5-infection, Mre11 is modified by SUMO-1 and SUMO-2/3 conjugation. Unexpectedly, SUMOylation of Mre11 is not exclusively dependent on E1B 55？kDa, E4 Orf6, or E4 Orf3, rather it seems to be influenced by a molecular interplay that involves each of these viral early proteins. 1. Introduction The adenoviral E1B 55？kDa, E4 Orf6, and E4 Orf3 early proteins are necessary to complete an efficient viral replication cycle. E1B 55？kDa and E4 Orf6 form a complex that associates with an adenovirus-infected cell-specific Cullin 5-containing E3 ubiquitin (Ub) ligase that induces polyubiquitination and degradation of various cellular targets, namely, the tumor suppressor p53, the Mre11-Rad50-Nbs1 (MRN) complex, DNA ligase IV, bloom helicase, ATRX, Tip60, SPOC1, and integrin α3 [1–12]. The activity of the E1B/E4 Orf6 E3-Ub ligase is required for viral late mRNA export [4, 13], and it has been suggested that most activities associated with E1B 55？kDa and E4 Orf6 depend on formation of this E3-Ub ligase [14]. However, during adenovirus infection of normal human cells (human foreskin fibroblasts, HFF), complete degradation of one of these cellular substrates, the p53 protein, is not attained and reduction of its steady state levels initiates only late during infection [15, 16]. Furthermore, the accumulation of p53 does not lead to apoptosis, and efficient viral replication correlates with the E1B 55？kDa-dependent nuclear localization of p53 to viral replication centers where these proteins colocalize [16–18]. An additional key target of the E1B/E4 Orf6 E3-Ub ligase is the DNA