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Journal of Virology, April 2001, p. 3859-3872, Vol. 75, No. 8
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.8.3859-3872.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Jin-Hyun Ahn,1,dagger Yixun Xu,2 Won-Jong Jang,2 Michael J. Matunis,3 and Gary S. Hayward1,2,*

Molecular Virology Program, Department of Oncology,1 and Department of Pharmacology and Molecular Sciences,2 Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, and Department of Biochemistry, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 212053

Received 9 October 2000/Accepted 19 January 2001

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.

* Corresponding author. Mailing address: Room 3M-10, Bunting-Blaustein Cancer Research Building, Oncology Center, Johns Hopkins University School of Medicine, 1650 Orleans St., Baltimore, MD 21231. Phone: (410) 955-8684. Fax: (410) 955-8685. E-mail: ghayward{at}jhmi.edu.

dagger Present address: Department of Molecular Cell Biology, Sungkyun Kwan University School of Medicine, Suwon 440-746, Korea.

Journal of Virology, April 2001, p. 3859-3872, Vol. 75, No. 8
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.8.3859-3872.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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