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 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.
Molecular Virology Program, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.
J H
JH
Ahn
Y
Y
Xu
W J
WJ
Jang
M J
MJ
Matunis
G S
GS
Hayward
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000096:biosynthesis; D006801:Humans; D017874:Immediate-Early Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D025842:SUMO-1 Protein; D016415:Sequence Alignment; D025841:Small Ubiquitin-Related Modifier Proteins; D013997:Time Factors; D015534:Trans-Activators; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014759:Viral Envelope Proteins; D014764:Viral Proteins; D014779:Virus Replication; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D004279:DNA, Viral; C088645:IE1 protein, cytomegalovirus; C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D008562:Membrane Glycoproteins; D025842:SUMO-1 Protein; C117909:SUMO2 protein, human; C492189:SUMO3 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D015534:Trans-Activators; C077406:UL115 protein, Human herpesvirus 5; D014452:Ubiquitins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C071440:glycoprotein H, Cytomegalovirus; C499358:glycoprotein H, Human cytomegalovirus; C439068:glycoprotein O, cytomegalovirus; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9