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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,
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.

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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