Evaluation of interactions of human cytomegalovirus immediate-early IE2 regulatory protein with small ubiquitin-like modifiers and their conjugation enzyme Ubc9

The human cytomegalovirus (HCMV) major immediate-early protein IE2 is a nuc lear phosphoprotein that is believed to be a key regulator in both lytic an d latent infections. Using yeast two-hybrid screening, small ubiquitin-like modifiers (SUMO-1, SUMO-2, and SUMO-3) and a SUMO-conjugating enzyme (Ubc9 ) were isolated as IE2-interacting proteins. In vitro binding assays with g lutathione S-transferase (GST) fusion proteins provided evidence for direct protein-protein interaction. Mapping data showed that the C-terminal end o f SUMO-1 is critical for interaction with IE2 in both yeast and in vitro bi nding assays, IE2 was efficiently modified by SUMO-1 or SUMO-2 in cotransfe cted cells and in cells infected with a recombinant adenovirus expressing H CMV IE2, although the level of modification was much lower in HCMV-infected cells. Two lysine residues at positions 175 and 180 were mapped as major a lternative SUMO-1 conjugation sites in both cotransfected cells and an in v itro sumoylation assay and could be conjugated by SUMO-1 simultaneously. Al though mutations of these lysine residues did not interfere with the POD (o r ND10) targeting of IE2, overexpression of SUMO-1 enhanced IE2-mediated tr ansactivation in a promoter-dependent manner in reporter assays. Interestin gly, many other cellular proteins identified as IE2 interaction partners in yeast two-hybrid assays also interact with SUMO-1, suggesting that either directly bound or covalently conjugated SUMO moieties may act as a bridge f or interactions between IE2 and other SUMO-1-modified or SUMO-1-interacting proteins. When we investigated the intracellular localization of SUMO-1 in HCMV-infected cells, the pattern changed from nuclear punctate to predomin antly nuclear diffuse in an IE1-dependent manner at very early times after infection, but with some SUMO-1 protein now associated with IE2 punctate do mains, However, at late times after infection, SUMO-1 was predominantly det ected,within viral DNA replication compartments containing IE2. Taken toget her, these results show that HCMV infection causes the redistribution of SU MO-1 and that IE2 both physically binds to and is covalently modified by SU MO moieties, suggesting possible modulation of both the function of SUMO-1 and protein-protein interactions of IE2 during HCMV infection.