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Journal of Virology, November 2001, p. 10683-10695, Vol. 75, No. 22
Molecular Virology Laboratories, Department
of Pharmacology and Molecular Sciences,1 and
Viral Oncology Program, Department of
Oncology,2 Johns Hopkins University School of
Medicine, Baltimore, Maryland 21231; Department of
Biochemistry and Molecular Biology,4 Johns
Hopkins University School of Hygiene and Public Health, Baltimore,
Maryland 21205; and Department of Molecular Cell
Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea3
Received 13 April 2001/Accepted 8 August 2001
Human cytomegalovirus (HCMV) major immediate-early protein IE1 is
an abundant 72-kDa nuclear phosphoprotein that is thought to
play an important role in efficient triggering of the lytic cycle,
especially at low multiplicity of infection. The best-known properties
of IE1 at present are its transient targeting to punctate promyelocytic leukemia protein (PML)-associated nuclear bodies (PML
oncogenic domains [PODs] or nuclear domain 10 [ND10]), with associated displacement of the cellular PML tumor suppressor protein into a diffuse nucleoplasmic form and its association with metaphase chromosomes. Recent studies have shown that the targeting of PML (and associated proteins such as hDaxx) to PODs is dependent on modification of PML by ubiquitin-like protein SUMO-1. In
this study, we provide direct evidence that IE1 is also covalently modified by SUMO-1 in both infected and cotransfected cells,
as well as in in vitro assays, with up to 30% of the protein
representing the covalently conjugated 90-kDa form in stable U373/IE1
cell lines. Lysine 450 was mapped as the major SUMO-1 conjugation site, but a point mutation of this lysine residue in IE1 did not interfere with its targeting to and disruption of the PODs. Surprisingly, unlike
PML or IE2, IE1 did not interact with either Ubc9 or SUMO-1 in yeast
two-hybrid assays, suggesting that some additional unknown intranuclear
cofactors must play a role in IE1 sumoylation. Interestingly, stable
expression of either exogenous PML or exogenous Flag-SUMO-1 in
U373 cell lines greatly enhanced both the levels and rate of in vivo
IE1 sumoylation during HCMV infection. Unlike the disruption of
PODs by the herpes simplex virus type 1 IE110(ICP0) protein, the
disruption of PODs by HCMV IE1 proved not to involve
proteasome-dependent degradation of PML. We also demonstrate here
that the 560-amino-acid PML1 isoform functions as a transcriptional
repressor when fused to the GAL4 DNA-binding domain and that wild-type
IE1 inhibits the repressor function of PML1 in transient
cotransfection assays. Furthermore, both IE1(1-346) and IE1(L174P)
mutants, which are defective in displacing PML from PODs, failed
to inhibit the repression activity of PML1, whereas the
sumoylation-negative IE1(K450R) mutant derepressed as
efficiently as wild-type IE1. Taken together, our results suggest that
proteasome-independent disruption of PODs, but not IE1 sumoylation,
is required for efficient IE1 inhibition of PML-mediated
transcriptional repression.
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.22.10683-10695.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Proteasome-Independent Disruption of PML Oncogenic Domains
(PODs), but Not Covalent Modification by SUMO-1, Is Required for Human
Cytomegalovirus Immediate-Early Protein IE1 To Inhibit PML-Mediated
Transcriptional Repression

*
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: Division of Bioinformatics, Science Applications
International Corporation, Arlington, VA 22203.
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