Evaluation of Interactions of Human Cytomegalovirus Immediate-Early IE2 Regulatory Protein with Small Ubiquitin-Like Modifiers and Their Conjugation Enzyme Ubc9

Abstract

The human cytomegalovirus (HCMV) major immediate-early protein IE2 is a nuclear phosphoprotein that is believed to be a key regulator in both lytic and 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 glutathione S-transferase (GST) fusion proteins provided evidence for direct protein-protein interaction. Mapping data showed that the C-terminal end of SUMO-1 is critical for interaction with IE2 in both yeast and in vitro binding assays. IE2 was efficiently modified by SUMO-1 or SUMO-2 in cotransfected cells and in cells infected with a recombinant adenovirus expressing HCMV 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 alternative SUMO-1 conjugation sites in both cotransfected cells and an in vitro sumoylation assay and could be conjugated by SUMO-1 simultaneously. Although mutations of these lysine residues did not interfere with the POD (or ND10) targeting of IE2, overexpression of SUMO-1 enhanced IE2-mediated transactivation in a promoter-dependent manner in reporter assays. Interestingly, 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 for 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 predominantly nuclear diffuse in an IE1-dependent manner at very early times after infection, but with some SUMO-1 protein now associated with IE2 punctate domains. However, at late times after infection, SUMO-1 was predominantly detected within viral DNA replication compartments containing IE2. Taken together, these results show that HCMV infection causes the redistribution of SUMO-1 and that IE2 both physically binds to and is covalently modified by SUMO moieties, suggesting possible modulation of both the function of SUMO-1 and protein-protein interactions of IE2 during HCMV infection. Human cytomegalovirus (HCMV) can cause severe disease complications and pathogenesis on infection of newborns or immunocompromised individuals, whereas infection of immunocompetent individuals is typically asymptomatic (8, 44).Gene expression during the permissive lytic cycle of HCMV occurs in a three-step sequential fashion. Shortly after infection, the immediate-early (IE) genes are expressed in the absence of de novo protein synthesis. IE proteins and virion factors are required for the subsequent induction of early and late genes (36, 56). The major IE (MIE) locus of HCMV genome encodes two nuclear phosphoproteins, namely, IE1 (UL123, IE72) and IE2 (UL122, IE86), which are translated from differentially spliced mRNA species (58, 60). Some a...

Members of the PIAS family act as SUMO ligases for c-Jun and p53 and repress p53 activity

“…We could show that PIASx␤ and PIAS1 do not stimulate transfer of SUMO to any substrate, suggesting that they indeed play a role in substrate selection. However, the fact that at least two substrates, p53 and c-Jun, are targeted by the same ligase and recent data reporting an interaction of PIAS1 with two other SUMO substrates, the androgen receptor (16,26) and the cytomegalovirus IE2 protein (27) may indicate that PIAS proteins are less important determinants for the substrate specificity in the SUMO pathway than are ubiquitin ligases in the ubiquitin pathway. However, an alternative possibility is that PIAS modifications or interactions with auxiliary factors may extend the repertoire of substrate specificity.…”

Human Herpesvirus 6 Immediate-Early 1 Protein Is a Sumoylated Nuclear Phosphoprotein Colocalizing with Promyelocytic Leukemia Protein-associated Nuclear Bodies

“…Work is in progress to address this issue. Sumoylation of herpesviruses IE proteins have also been reported for HCMV and Epstein-Barr virus (36,47,48,73,74). The effects of SUMO conjugation on the functionality of HHV-6 IE1 remain unknown.…”

“…SUMO conjugation is reported to affect proteins function in a variety of different ways. For example, the ability of the p53 and HCMV IE2 proteins in promoting transcriptional activation is positively influenced by SUMO conjugation (73)(74)(75)(76). Whether a similar activity can be expected for IE1 of HHV-6 remains to be determined.…”

SUMO: of branched proteins and nuclear bodies

“…Later work extended these ®ndings by showing that some immediate-early viral gene products are themselves substrates for sumoylation. These include CMV-IE1 (MuÈ ller and Dejean, 1999), and -IE2 (Ahn et al, 2001;Hofmann et al, 2000), bovine and human papillomavirus (BPV, HPV) E1 (Rangasamy et al, 2000) and the EBV-BZFL1 (Adamson and Kenney, 2001). The signi®cance here of sumoylation remains poorly understood, but for BPV-E1 it appears necessary for normal nuclear localization, whereas for CMV-IE2, sumoylation was shown to enhance IE2-mediated transactivation.…”