SUMOylation of the human cytomegalovirus 72-kilodalton IE1 protein facilitates expression of the 86-kilodalton IE2 protein and promotes viral replication.

JOURNAL OF VIROLOGY, July 2004, p. 7803–7812 Vol. 78, No. 14
0022-538X/04/$08.00�0 DOI: 10.1128/JVI.78.14.7803–7812.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
SUMOylation of the Human Cytomegalovirus 72-Kilodalton IE1
Protein Facilitates Expression of the 86-Kilodalton IE2
Protein and Promotes Viral Replication
Michael Nevels,† Wolfram Brune,‡ and Thomas Shenk*
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014
Received 5 November 2003/Accepted 6 April 2004
The 72-kDa immediate-early 1 protein (IE1-72kDa) of human cytomegalovirus has been previously shown to
be posttranslationally modified by covalent conjugation to the ubiquitin-related protein SUMO-1. Using an
infectious bacterial artificial chromosome clone of human cytomegalovirus, we constructed a mutant virus
(BADpmIE1-K450R) that is deficient for SUMOylation of IE1-72kDa due to a single amino acid exchange in
the SUMO-1 attachment site. Compared to wild-type virus, this mutant grew more slowly and generated a
reduced yield in infected human fibroblasts, indicating that SUMO modification is required for the full activity
of IE1-72kDa. The lack of SUMOylation did not affect the intranuclear localization of IE1-72kDa, including its
ability to target to and disrupt PML bodies and to bind to mitotic chromatin. Likewise, SUMOylation-deficient
IE1-72kDa activated several viral promoters as efficiently as the wild-type protein. However, the failure to
modify IE1-72kDa resulted in substantially reduced levels of the IE2 transcript and its 86-kDa protein
(IE2-86kDa). These observations suggest that SUMO modification of IE1-72kDa contributes to efficient HCMV
replication by promoting the accumulation of IE2-86kDa.
Covalent attachment of ubiquitin (ubiquitylation) has long
been known to serve as a tag for protein degradation via the
26S proteasome (reviewed in reference 59). Additionally, a num-
ber of ubiquitin-related proteins have been recently identified
that are joined to other proteins through an enzymatic process
that is biochemically analogous to, but functionally distinct from,
ubiquitylation. Among the best characterized of these proteins
are the small ubiquitin-like modifiers SUMO-1 (SMT3C),
SUMO-2 (SMT3A), and SUMO-3 (SMT3B) (reviewed in ref-
erences 29 and 60). While SUMOylation does not typically
target proteins for degradation, it can have diverse effects on
its substrates. SUMO modification enhances the stability of
some cellular proteins (9, 16). In other cases, it modulates the
subcellular localization and/or transactivation properties of its
target proteins (reviewed in references 48, 67, and 71).
The list of substrates for SUMOylation includes an increas-
ing number of viral regulatory proteins (reviewed in reference
72). Protein products of bovine papillomavirus E1 (51, 52),
vaccinia virus E3L (56), adenovirus type 5 E1B (18), human
herpesvirus 6 IE1 (21), and Epstein-Barr virus BZLF1 (1), as
well as the major immediate-early proteins of human cytomeg-
alovirus (HCMV), the 72-kDa immediate-early 1 protein (IE1-
72kDa) and the 86-kDa immediate-early 2 protein (IE2-
86kDa) (5, 26, 44, 63, 73), have all been shown to be targets of
SUMO-1 conjugation. In addition, HCMV IE2-86kDa is mod-
ified by SUMO-2 and SUMO-3 (5, 26). While the functional
consequences of SUMOylation are unknown for most of these
viral proteins, transfection assays indicate that SUMO-1 mod-
ification is critical for nuclear import of the papillomavirus E1
protein (52) and for the nuclear accumulation, intranuclear tar-
geting, and transforming functions of adenovirus E1B-55kDa
(18). In contrast, SUMOylation does not obviously affect the
subcellular localization of HCMV IE2-86kDa but enhances its
transactivation capacity (5, 26). The biological consequences of
SUMOylation for the life cycles of the respective viruses, how-
ever, remain to be determined, since in none of these cases has
the SUMOylation site been mutated in the viral genome to allow
for the study of this modification in the context of virus infection.
The UL123-coded IE1-72kDa and UL122-coded IE2-86kDa
are abundant nuclear phosphoproteins expressed from alter-
natively spliced transcripts that originate from the major im-
mediate-early locus of HCMV (reviewed in references 13 and
42). They share 85 amino-terminal amino acids corresponding
to major immediate-early exons 2 and 3 but have distinct car-
boxy-terminal parts encoded by exon 4 (IE1) or exon 5 (IE2).
Both proteins are believed to be important transcriptional reg-
ulators. IE2-86kDa interacts with multiple components of the
cellular transcription machinery, promiscuously activating a
wide range of viral and cellular promoters. Consistent with a
role as the principal transcriptional activator of the HCMV
lytic cycle, IE2-86kDa has been shown to be essential for early
viral gene expression and productive viral growth in tissue
culture (24, 38).
In transient-transfection assays IE1-72kDa can modestly
augment transcription from a number of viral and cellular
promoters, including the HCMV major immediate-early pro-
moter, various HCMV early gene promoters, the SV40 pro-
moter (reviewed in references 13 and 42), and the promoter of
the human origin recognition complex 1 (hOrc-1) gene (62).
Moreover, coexpression of IE1-72kDa can boost the transac-
* Corresponding author. Mailing address: Department of Molecular
Biology, Princeton University, Princeton, NJ 08544-1014. Phone: (609)
258-5992. Fax: (609) 258-1704. E-mail: tshenk@princeton.edu.
† Present address: Institut fu¨r Medizinische Mikrobiologie und Hy-
giene, Universita¨t Regensburg, Forschungszentrum (FZL), D-93047
Regensburg, Germany.
‡ Present address: Rudolf-Virchow-Zentrum fu¨r Experimentelle
Biomedizin, Universita¨t Wu¨rzburg, D-97078 Wu¨rzburg, Germany.
7803

tivation capacity of IE2-86kDa. IE1-72kDa is not believed to
bind DNA directly but physically interacts with several cellular
transcription factors and other nuclear proteins, including
CTF-1 (23), Sp-1 (76), E2F1 to -5 (39), TAFII130 (37), p107
(49), Daxx (D. L. Woodhall, L. A. Teague, G. W. Wilkinson, S.
Efstathiou, and J. H. Sinclair, presented at the 28th Interna-
tional Herpesvirus Workshop, Madison, Wis., 2003), and PML
(2). The last two are key components of nuclear multiprotein
complexes known by several names: PML bodies, PML onco-
genic domains, or nuclear domain 10s (ND10s). ND10s have
been implicated as sites for input viral genome deposition as
well as for immediate-early transcription and initiation of viral
DNA replication in HCMV and a number of other DNA
viruses (reviewed in references 19 and 55). IE1-72kDa is
known to be necessary and sufficient to disrupt ND10s during
the early stages of HCMV infection and in transfected cells (4,
28, 31, 70). Intriguingly, IE1-72kDa can interact not only with
the interchromatinic ND10 structures but also with chromatin,
which results in colocalization with chromosomes during mi-
tosis (2, 32, 70). The significance of these interactions with
higher-order structures of the host cell nucleus is largely un-
known, but it is tempting to speculate that they are linked to
the transactivating properties of IE1-72kDa. Alternatively,
they may be related to one or more of the other activities that
have been ascribed to this viral protein. Specifically, IE1-
72kDa has been shown to stimulate viral DNA replication (47,
58), affect cell cycle progression (14), block apoptosis (75, 79),
cotransform cells to an oncogenic phenotype, and exhibit mu-
tagenic activity (61). IE1-72kDa also displays kinase activity
(46), and it is a dominant target for cell lysis by cytolytic CD8�
T lymphocytes (reviewed in reference 53). The various activi-
ties that have been attributed to IE1-72kDa do not yet show a
clear picture of this protein’s role in the context of the viral life
cycle. However, IE1-deficient mutant viruses display dimin-
ished replication efficiency, a decreased ability to form plaques,
an inability to generate intranuclear replication compartments,
and a broad defect in expression of viral early genes (20, 22,
43). This phenotype is especially pronounced when cells are
infected at a low multiplicity of infection. Taken together,
these observations suggest that IE1-72kDa is a pleiotropic reg-
ulator of the early events in the lytic infectious cycle whose
functions are modulated by posttranslational modifications as
well as noncovalent and covalent protein interactions.
Previous work has demonstrated that SUMO-1 conjugation
occurs at lysine residue 450 (K450) within a SUMOylation
consensus sequence in IE1-72kDa (63, 73). However, no func-
tional consequences of this modification have been revealed
(44, 63, 73). As is the case for all other known SUMOylated
viral proteins, no recombinant virus with a specific mutation of
the SUMOylation site in IE1-72kDa has been reported. Using
an infectious bacterial artificial chromosome (BAC) clone of
HCMV, we constructed a mutant (pBADpmIE1-K450R) that
is deficient for SUMOylation of IE1-72kDa due to a single
amino acid exchange in the SUMO-1 attachment site, and we
describe the mutation’s phenotypic consequences.
MATERIALS AND METHODS
Cloning of IE1 expression plasmids. The plasmid pCGN-IE1 expresses
HCMV IE1-72kDa fused to an amino-terminal influenza virus hemagglutinin
(HA) epitope tag under control of the HCMV major immediate-early promoter-
enhancer (79). The plasmid pCGN-IE1-K450R was derived from pCGN-IE1 by
site-directed mutagenesis with the QuikChange procedure (Stratagene) accord-
ing to the manufacturer’s instructions. The oligonucleotides used for mutagen-
esis were K450R-fw (5�-GACACTGTGTCTGTCCGGTCTGAGCCAGTGTCT
G-3�) and K450R-rv (5�-CAGACACTGGCTCAGACCGGACAGACACAGT
GTC-3�). Error-free mutagenesis was verified by DNA sequence analysis of the
entire IE1-72kDa-coding region.
For the construction of pEGFP-IE1, which expresses wild-type IE1-72kDa
fused to the carboxy terminus of the enhanced green fluorescent protein
(EGFP), the IE1-coding sequence was released from pCGN-IE1 with KpnI and
BamHI and inserted into the same sites of pcDNA3 (Invitrogen). From the
resulting plasmid, pcDNA-IE1, the insert was subsequently excised with HindIII
and BamHI and inserted in frame into the HindIII and BamHI sites of
pEGFP-C1 (Clontech). To generate plasmid pEGFP-IE1-K450R, which ex-
presses the respective IE1-72kDa mutant fused to EGFP, pEGFP-C1 was cut
with SacI and BamHI and ligated with the SacI-BamHI fragment from pCGN-
IE1-K450R.
Mutagenesis of HCMV genomes. HCMV mutants were produced by using a
BAC clone of the HCMV AD169 genome (AD169-BAC [25], referred to as
pBADwt in this publication). They were generated by homologous recombina-
tion in Escherichia coli with linear, PCR-generated DNA fragments (74, 77;
reviewed in references 12, 40, and 68).
For the generation of the IE1 knockout deletion mutant BADsubIE1, an
ampicillin resistance (amp) gene was excised from vector pST76A (50) by using
the restriction enzymes EcoRI and NotI and inserted into the MfeI and NotI
sites of vector pECFP-N1 (Clontech), downstream of the enhanced cyan fluo-
rescent protein (ECFP) gene. The resulting plasmid, pECFP-amp, served as a
template for PCR amplification of the ECFP-coding sequence linked to the amp
cassette by using purified primers that contained 50 nucleotides for homologous
recombination with the DNA sequences flanking IE1 exon 4 in addition to 20 or
21 nucleotides for hybridization with the PCR template (IE1-ECFP-amp-fw,
5�-AGGAGGACGGATACTTATATGTGTTGTTATCCTCCTCTACAGTCA
AACAGAATTCGGTGAGCAAGGGCGAGGAGCTG-3�; IE1-ECFP-amp-rv,
5�-GTGACGTGGGATCCATAACAGTAACTGATATATATATATACAATA
GTTTAGCAAGTGGCACTTTTCGGGG-3�). Additionally, an EcoRI site was
introduced into primer IE1-ECFP-amp-fw to facilitate identification of recom-
binant BACs by restriction digestion. Allelic exchange of BACs with linear DNA
fragments was carried out in E. coli strain DY380, which expresses the recom-
bination genes exo, bet, and gam of bacteriophage � in a temperature-dependent
fashion, as described in detail elsewhere (34, 74). BAC DNAs from a number of
ampicillin-resistant colonies were prepared, digested with different restriction
enzymes, and separated on 0.6% agarose gels to verify the success of the recom-
bination procedure and the overall integrity of the viral genome. The accuracy of
mutagenesis was further confirmed by PCR and DNA sequence analyses.
For the construction of the IE1 point mutant BAC pBADpmIE1-K450R and
the revertant pBADrevIE1, a BamHI site was introduced into plasmid pCGN-
IE1 directly after the stop codon of the IE1-coding sequence by using the
QuikChange mutagenesis strategy (primers were IE1-B-fw [5�-GCAAGGCTG
ACCAGTAAGGATCCGTATATATATATCAG-3�] and IE1-B-rv [5�-CTGAT
ATATATATACGGATCCTTACTGGTCAGCCTTGC-3�]) to generate pCGN-
IE1-B. Likewise, one EcoRI site was converted into a BamHI site in plasmid
pSLFRTKn (6) by using primers FKF-B-fw (5�-CGTCGTGGAATGCCTTCG
GATCCGAAGTTCCTATACTTTC-3�) and FKF-B-rv (5�-GAAAGTATAGG
AACTTCGGATCCGAAGGCATTCCACGACG-3�). This was followed by ex-
cision of the kanamycin resistance (kan) gene flanked by FLP recognition target
(FRT) sites from this plasmid with BamHI and insertion into the BamHI site of
pCGN-IE1-B. The resulting plasmid (pCGN-IE1-B-kan) was used as a template
for QuikChange mutagenesis with primers K450R-fw and K450R-rv. This pro-
cedure resulted in plasmid pCGN-IE1-K450R-B-kan, containing two nucleotide
substitutions in the IE1-coding sequence, i.e., AAG to CGG in codon 450. All
mutations were verified by comprehensive DNA sequence analysis. Fragments
comprising the mutated IE1 sequences linked to the FRT-flanked kan gene were
PCR amplified by using templates pCGN-IE1-B-kan and pCGN-IE1-K450R-B-
kan together with primers IE1-Kan-fw (5�-AGGAGGACEGATACTTATATG
TGTTGTTATCCTCCTCTACAGTCAAACAGATTAAGGTTCGAGTGG-
3�) and IE1-Kan-rv (5�-GTGACGTGGGATCCATAACAGTAACTGATATAT
ATATATACAATAGTTTAAGGACGACGACGACAAGTAA-3�). Again, these
oligonucleotides contained an additional 50 nucleotides for homologous recombi-
nation. Linear recombination with the purified PCR products and pBADsubIE1 in
E. coli DY380 was performed as described above, and the integrity of BAC DNAs
(pBADpmIE1-K450R-kan and pBADrevIE1-kan) from ampicillin-sensitive, kana-
mycin-resistant bacterial colonies was confirmed by restriction digestion. DNAs from
selected BAC clones were subsequently transformed into E. coli strain DH10B, and
7804 NEVELS ET AL. J. VIROL.

kan cassettes were removed by FRT site-directed recombination with FLP
recombinase expressed from plasmid pCP20 (15). This plasmid replicates in
a temperature-dependent fashion, allowing FLP-mediated recombination at
30°C (permissive temperature) and its own elimination at 42°C (restrictive
temperature). Single colonies were screened for kanamycin sensitivity, and
BACs from selected colonies were further analyzed by restriction digestion
and DNA sequence analyses.
Cell culture and virus infections. The following cell types were used in this
study: primary human foreskin fibroblasts (passages 5 to 18), ihf-2 cells (a
fibroblast cell line that was immortalized by introduction of the human papillo-
mavirus E6 and E7 genes) (22), ihfie1.3 cells (a gift from E. Mocarski, Stanford
University), E6/E7-immortalized fibroblasts that stably express HCMV IE1-
72kDa (43), and the human non-small-cell lung carcinoma cell line H1299 (41).
All cells were cultured as monolayers in medium containing 10% fetal calf serum
at 37°C.
HCMV was grown and titers were determined on fibroblasts by standard
procedures. For viral growth analyses, infected cells and culture medium were
combined and subjected to one freeze-thaw cycle followed by a short sonication
step, and virus titers were determined according to the median tissue culture
infectious dose method (54) on fibroblasts (BADwt and BADrevIE1) or ihfie1.3
cells (BADpmIE1-K450R). To reconstitute infectious virus from wild-type and
mutant viral genomes, BAC DNA was purified by using the NucleoBond plasmid
kit (BD Biosciences Clontech), and 2 �g of this DNA was transfected into
fibroblasts by electroporation as previously described (7). Along with the BAC
DNA, 1 �g of pCGN-pp71, which enhances the infectivity of HCMV DNA (7),
and 1 �g of the cre-expressing plasmid pBRep-Cre (25), to direct excision of the
BAC sequences from the viral genome, were included in the transfection mix.
Fluorescence microscopy. For indirect immunofluorescence analyses, subcon-
fluent fibroblasts received 5 �g of plasmid DNA by electroporation as previously
described (7). Following transfection, cells were plated on coverslips. Alterna-
tively, cells were infected with BADpmIE1-K450R-1 or BADwt at a multiplicity
of 3 or 0.01 PFU/cell. At different times postinfection, cells were washed twice
with phosphate-buffered saline (PBS) and fixed with paraformaldehyde (2% in
PBS) for 15 min at room temperature, followed by three 5-min washes with PBS
and permeabilization with 0.1% Triton X-100 in PBS for 15 min. After three
washes in PBS the permeabilized cells were blocked for 1 h in PBS containing 2%
bovine serum albumin and 0.05% Tween 20. After that, samples were washed
once in PBS and reacted for 1 h at room temperature with the appropriate
primary antibodies in a humidity chamber, followed by three 5-min washes with
PBS and a 1-h incubation with the appropriate Alexa 488- or Alexa 546-conju-
gated secondary antibody (Molecular Probes). After three additional washes in
PBS, coverslips were mounted in slow-fade solution (Molecular Probes), and
images were acquired on a Nikon TE200 fluorescence microscope with a charge-
coupled device camera (Diagnostic Instruments) or a Zeiss LSM laser scanning
microscope. The primary antibodies used in this study were rat anti-HA purified
monoclonal antibody (3F10; Roche), mouse anti-IE1 monoclonal hybridoma
supernatant (1B12) (79), mouse anti-PML monoclonal hybridoma supernatant
(5E10) (65), and rabbit anti-Sp100 polyclonal serum (AB1380; Chemicon).
For live-cell visualization of EGFP fusion proteins, H1299 cells were grown on
glass-bottom dishes (MatTek), and approximately 50% confluent cultures were
transfected with 5 �g of plasmid pEGFP-IE1 or pEGFP-IE1-K450R by using a
modified calcium phosphate precipitation procedure. At about 48 h after trans-
fection, cells were stained with Hoechst 33342 according to the manufacturer’s
instructions, and confocal images were acquired on a Zeiss LSM 510 laser
scanning microscope. ECFP expressed from recombinant virus BADsubIE1-1
was detected 3 days after transfection of the respective BAC DNA (2 �g) into
fibroblasts by electroporation (7).
Reporter gene assays. For luciferase assays, subconfluent fibroblast monolay-
ers were transfected by using the Fugene 6 reagent (Roche) with 1 �g of reporter
and 0.1 �g of effector plasmid DNA according to the manufacturer’s instructions.
Plasmid pCGN-IE2 expresses HCMV IE2-86kDa under control of the HCMV
major immediate-early promoter-enhancer and has been previously described
(79). The following reporter plasmids were used in this study: pGL3-Promoter
(Promega), pGL3-ICP36 (57), pGL3-MIEP (57), and pHsOrc1Luc (62). Infec-
tions (multiplicity of infection [MOI] � 3 PFU/cell) were performed 24 h after
transfection of reporter plasmids, and cells were collected at 18 h postinfection.
Western blot analyses. To assay the accumulation of viral proteins in HCMV-
infected cells, lysates were prepared at various times after infection by suspend-
ing equal amounts of cells directly in 2� sample buffer (100 mM Tris [pH 6.8],
200 mM dithiothreitol, 4% sodium dodecyl sulfate, 0.2% bromophenol blue,
20% glycerol, 40 �M N-ethylmaleimide), followed by heating of the samples at
95°C for 10 min, quick sonication, and additional heating at 95°C for 10 min.
Aliquots were separated in sodium dodecyl sulfate-containing 10% polyacryl-
amide gels, and the proteins were transferred to nitrocellulose membranes.
Nonspecific binding was blocked by incubation in PBS containing 5% nonfat dry
milk for at least 2 h. After that, membranes were washed once in PBS and probed
with primary mouse monoclonal antibodies specific for the following viral or
cellular proteins for 1 to 12 h at room temperature or 4°C, respectively: �-tubulin
(DM1A; Sigma), IE1 (1B12) (79), IE1/IE2 (MAB810; Covance), SUMO-1
(21C7; Zymed), and ppUL44 (anti-ICP36; Virusys). After three washes with PBS
containing 0.1% Tween 20, membranes were incubated with anti-mouse immu-
noglobulins conjugated with horseradish peroxidase (Dako) for 1 h, followed by
three washes with PBS–0.1% Tween 20 and chemiluminescence detection (Super
Signal; Pierce).
Northern blot hybridization. Cells on 15-cm-diameter dishes were scraped
into 10 ml of Trizol reagent (Gibco Life Sciences) at the indicated times after
infection and stored at �80°C. After completion of the time course, samples
were thawed, and RNA was purified by using the Trizol protocol. Then, 10 �g of
each purified RNA preparation was electrophoretically separated on formalde-
hyde gels and blotted to Hybond N� membranes (Amersham). DNA probes
were generated and nonradioactively labeled with digoxigenin-11-dUTP (Roche)
by PCR with the indicated cDNAs (IE1 exon 4, IE2 exon 5, UL37 exon 1, TRS1,
and UL44) in pGEM-T (Promega) (11) as templates and universal primers for
the T7 and Sp6 promoter sequences. Membranes were hybridized to the labeled
probes overnight at 42°C in DIG Easy Hyb buffer (Roche). Washing and anti-
body detection were performed as recommended by the manufacturer.
RESULTS
Construction of HCMV BACs with mutations in the IE1
gene. It has been previously demonstrated that posttransla-
tional modification of IE1-72kDa by conjugation to SUMO-1
occurs at a single acceptor site (K450), and mutation of K450
to arginine (K450R) results in loss of IE1-72kDa SUMOyla-
tion in transfected cells and in cell-free assays (63, 73). Based
on these observations, we employed the BAC system to intro-
duce two nucleotide exchanges into the viral genome that re-
sult in the expression of a K450R mutant IE1-72kDa. A three-
step strategy was employed, which allows for the introduction
of any point mutation into the viral BAC without requiring the
presence of restriction enzyme sites, further cloning, or shuttle
plasmids. Instead, the first two recombination steps were per-
formed by allelic exchange with linear, PCR-generated frag-
ments, and the third step involved site-specific excision by the
FLP recombinase. To our knowledge, this three-step method
has not been applied before to the mutagenesis of viral genomes.
In step one, we generated three mutant BAC clones
(pBADsubIE1-1 to -3) in which almost all of the IE1-specific
exon 4 sequence (except the nucleotides encoding the first
three amino acids and the stop codon) was replaced by an amp
gene and an ECFP-coding sequence (Fig. 1A). The ECFP
sequence was introduced to facilitate the identification of in-
fected cells by fluorescence microscopy and was inserted in a
way that permits splicing to major immediate-early exon 3 and
in-frame translation as a fusion protein comprising an 85-
residue amino-terminal peptide (expressed from major imme-
diate-early exons 2 and 3) and the fluorescent protein. Accord-
ingly, after transfection of pBADsubIE1 DNAs into
fibroblasts, transfected cells displayed predominantly nuclear
fluorescence (data not shown), consistent with the earlier pro-
posal that amino acids 1 to 24 of IE1-72kDa comprise a nu-
clear localization signal (70). The identity and integrity of the
mutant BACs were further confirmed by restriction digestion
with different enzymes (Fig. 1B and data not shown) and PCR
analysis (Fig. 1C).
In step two, linear recombination was used to replace the
VOL. 78, 2004 MUTANT HCMV DEFICIENT FOR SUMOylation OF IE1-72kDa 7805

ECFP-amp cassette in pBADsubIE1-1 with an IE1 exon 4
sequence that was modified to contain the desired nucleotide
substitutions corresponding to the K450R amino acid change
(Fig. 2A). Attached to the mutated IE1 gene, a kan cassette
that was flanked by FRT sites was inserted, resulting in
pBADpmIE1-K450R-kan. In step 3, the FRT sites allowed for
subsequent excision of the kan gene by expression of the site-
specific FLP recombinase, thus leaving only viral sequences at
the mutation site and a small insertion of 54 nucleotides cor-
responding to a single FRT site (34 bp) plus the primer binding
site (20 bp) used for generating the PCR fragments (Fig. 2A).
Following this three-step procedure, two independent clones
of pBADpmK450R were generated. To control for potential
functional effects of the 54 nucleotide insertion, two revertant
BACs (pBADrevIE1) were constructed from pBADsubIE1-1
by using the wild-type IE1 sequence for allelic exchange.
Again, the identity and integrity of all recombinant BACs were
verified by various restriction digestions and DNA sequence
analysis (Fig. 2B and data not shown).
Infectivity of mutant BACs. We electroporated purified
pBADwt, pBADsubIE1, pBADpmIE1-K450R, and pBADrevIE1
DNAs into permissive fibroblasts and monitored the trans-
fected cultures for plaque development due to reconstituted
FIG. 1. Generation and characterization of pBADsubIE1 mutant
BACs. (A) Introduction of a targeted substitution mutation. The po-
sitions of restriction enzyme sites used for cloning are indicated by
capital letters (E, EcoRI; M, MfeI; N, NotI). Black block arrows,
ECFP, amp, and major immediate-early exon 4 or exon 3 DNA se-
quences, with the arrowheads pointing in the sense-strand directions;
hatched segments, introns that flank the IE1-specific exon 4, where the
crossover events (indicated by crossed lines) during homologous re-
combination in E. coli occurred; narrow arrows, PCR primers. Ele-
ments in this diagram are not drawn to scale. (B) Restriction digest
with EcoRI of DNA from the wild-type HCMV BAC and three inde-
pendent mutant BAC clones. In all three mutants, due to an EcoRI site
that was recombined with the ECFP-amp cassette, the 10,026-bp band
(arrowhead) present in the wild-type disappears. Instead, two bands of
8,035 bp (arrowhead) and 3,306 bp (not visible) appear. Markers are a
1-kb ladder. (C) PCR analysis with the wild-type BAC and one of the
mutant BAC clones or without template (H2O). PCR primers are
designated a, b, and c (a, IEP3C; b, IEP4J; c, IEP5B) (30), and their
approximate positions within the HCMV major immediate-early re-
gion are indicated by arrowheads. Boxes represent exons of the
HCMV major immediate-early region (the numbers 1 to 5 correspond
to major immediate-early exons 1 to 5) or the ECFP and amp genes
(coding sequences are shown in black, and noncoding sequences are
shown in white). Lines represent introns or other noncoding se-
quences. An ethidium bromide-stained 1% agarose gel shows PCR
products of the expected sizes amplified with the indicated primer
combinations and templates (lane 4, 1,158 bp; lane 5, 1,786 bp; lane 7,
3,105 bp). As predicted, the reactions in lanes 2, 3, and 6 did not result
in amplification products. Markers (lane 1) are a 1-kb ladder.
FIG. 2. Construction and analysis of IE1 point mutant BACs and
viruses. (A) Diagram showing the recombination strategy that was
used to generate pBADpmIE1-K450R from pBADsubIE1. Boxes with
numbers represent major immediate-early exons 1 to 5. The ECFP,
amp, and kan genes are also shown as boxes. Open triangles symbolize
specific point mutations, and FRT sites are represented by small white
boxes with the letter F inside (coding sequences are shown in black,
and noncoding sequences are shown in white). Lines represent introns
or other noncoding sequences. (B) Restriction digests of wild-type (wt,
pBADwt) and mutant (K450R, pBADpmIE1-K450R; rev, pBADrevIE1)
BAC DNAs (two independently generated clones per mutant are des-
ignated with the suffix -1 or -2) with EcoRI followed by electrophoresis
in a 0.6% ethidium bromide-stained agarose gel. All BAC preparations
show a wild-type pattern of restriction fragments. Markers are a 1-kb
ladder. (C) Western blot analysis with anti-IE1 antibody 1B12 and
whole-cell lysates from uninfected cells (Mock) or fibroblasts infected
with BADwt (wt), BADpmIE1-K450R-1 (K450R-1), and BADrevIE1-1
(rev-1) at an MOI of 3 PFU/cell. The blots show bands corresponding
to the SUMOylated and/or nonconjugated IE1-72kDa at 5 or 20 h
postinfection (hpi).
7806 NEVELS ET AL. J. VIROL.

virus. The transfection experiments were repeated multiple
times with several independently generated BAC clones and
preparations to minimize the chance that negative results were
due to deficiencies in the integrity or quality of BAC DNAs.
After 7 to 10 days, nascent plaques could be identified, and at
14 days after transfection, infectious virus was quantified from
the supernatant of the cultures. In the four representative
experiments shown in Table 1, and in several additional trans-
fections, the pBADwt, pBADpmIE1-K450R, and pBADrevIE1
clones consistently yielded infectious virus, although the titers
of the reconstituted wild-type and revertant viruses were about
10-fold higher than those of the K450R mutants. In contrast,
pBADsubIE1 never generated detectable virus. The failure to
recover infectious virus from these transfections argues that a
functional IE1 gene is essential for the full infectivity of
HCMV BAC DNA. Moreover, these experiments show that
SUMO modification is not absolutely required for the function
of IE1-72kDa during lytic infection.
K450R mutant viruses display attenuated growth. After vi-
rus reconstitution from pBADwt, pBADpmIE1-K450R, and
pBADrevIE1, virus stocks for which the titers were determined
were prepared, and the lack of IE1-72kDa SUMOylation in the
K450R mutant virus was confirmed by Western blotting with
an IE1-specific monoclonal antibody (Fig. 2C). Even after
overexposure of the protein blot, no 92-kDa high-molecular-
mass band, which has been shown previously to correspond to
SUMOylated IE1-72kDa (44, 63, 73), was present in the
K450R mutant under conditions where this species was easily
detected in cells infected with the wild-type and revertant vi-
ruses (Fig. 2C). Likewise, an antibody directed against
SUMO-1 could not detect any modified IE1 protein in cells
infected with BADpmIE1-K450R-1 (data not shown).
Subsequently, single-step (multiplicity of 3 PFU/cell) and
multistep (multiplicity of 0.01 PFU/cell) growth analyses were
performed. The single-step analyses were done both with com-
plementing, immortalized fibroblasts that stably express wild-
type IE1-72kDa (ihfie1.3 cells) (43) and with noncomplement-
ing parental cells (ihf-2 cells) (22). For the multistep growth
curves, primary fibroblasts were used. Compared to the wild-
type and revertant viruses, the IE1-K450R mutant displayed
slower replication at both low and high MOIs, with an about
10-fold difference in virus titers on noncomplementing cells
(Fig. 3A and B). These observations are consistent with the
results obtained by quantitation of reconstituted virus after
BAC transfection (Table 1). Importantly, the K450R-specific
growth defect was almost completely offset in cells exogenously
expressing IE1-72kDa (Fig. 3C). Moreover, in the multistep
experiment, similar growth defects could be detected with mu-
tant viruses derived from two independent pBADpmIE1-K450R
clones, while two different revertant viruses (BADrevIE1)
showed growth kinetics that were very similar to those of the
wild type (Fig. 3A). These results indicate that the observed
FIG. 3. Growth characteristics of SUMOylation-deficient IE1 mu-
tant HCMVs compared to wild-type and revertant viruses. Symbols
represent average values from two experiments performed in parallel.
Open circles, BADwt; open triangles, BADrevIE1-1; open squares,
BADrevIE1-2; filled squares, BADpmIE1-K450R-1; filled triangles,
BADpmIE1-K450R-2. (A) Growth kinetics after low-multiplicity
infection (0.01 PFU/cell) of fibroblasts. (B) Growth kinetics after
high-multiplicity infection (3 PFU/cell) of ihf-2 cells. (C) Growth
kinetics after high-multiplicity infection (3 PFU/cell) of complement-
ing ihfie1.3 cells.
TABLE 1. Virus titers at 20 days after transfection of fibroblasts
with HCMV BAC constructsa
BAC construct
Virus titer (median tissue culture infective dose)
in expt:
1 2 3 4
pBADwt 5.4 � 106 6.8 � 106 3.2 � 106 5.3 � 106
pBADsubIE1-1 0 NDb 0 0
pBADsubIE1-2 0 0 ND ND
pBADsubIE1-3 0 ND 0 ND
pBADpmIE1-K450R-1 5.0 � 105 5.2 � 105 4.4 � 105 2.8 � 105
pBADpmIE1-K450R-2 3.1 � 105 1.0 � 105 ND ND
pBADrevIE1-1 4.7 � 106 4.2 � 106 6.1 � 106 3.0 � 106
pBADrevIE1-2 7.9 � 106 2.2 � 106 ND ND
a Multiple independently generated mutant clones were tested.
b ND, not determined.
VOL. 78, 2004 MUTANT HCMV DEFICIENT FOR SUMOylation OF IE1-72kDa 7807

phenotype of the K450R mutant viruses is a specific effect due
to the point mutation in IE1-72kDa, demonstrating that
SUMOylation of this viral protein promotes efficient virus rep-
lication in fibroblasts.
Subcellular localizations and nuclear interactions of the
IE1-72kDa mutant proteins. Previous work has shown that
modification by SUMO-1 is critical for the (sub)nuclear tar-
geting of some viral proteins (18, 52), and SUMOylation of the
cellular PML protein is known to be important for its associ-
ation with ND10s (17, 33, 45, 78). IE1-72kDa is transiently
colocalized with PML at ND10s in the first hours after infec-
tion, and subsequently the two proteins are redistributed, dis-
playing a uniform nuclear diffuse pattern (4, 28, 31, 70). Thus,
to investigate how SUMO conjugation contributes to the func-
tion of IE1-72kDa, we asked whether the SUMOylation-defi-
cient K450R mutant protein can still target to the nucleus and
disrupt ND10s as efficiently as wild-type IE1-72kDa. For this
purpose, we transfected fibroblasts with plasmids expressing
wild-type IE1-72kDa and IE1-K450R and performed double-
label immunofluorescence analyses with antibodies against the
IE1-72kDa and PML proteins. We found that IE1-K450R was
still able to efficiently target to the nucleus, localizing in a
diffuse pattern that was indistinguishable from the wild-type
pattern, indicating that the mutant protein retained the ability
to trigger the redistribution of PML (Fig. 4A). These observa-
tions confirm results of previous studies that have been carried
out with transfected cells (44, 63, 73).
To verify these observations in the context of an HCMV
infection, we exposed fibroblasts to equal amounts of the wild-
type and K450R mutant viruses (MOI � 3 PFU/cell) and
monitored the temporal subnuclear distribution patterns of
IE1-72kDa and Sp100, an alternative marker for ND10s (66),
over a time course of 24 h. As expected, at 1 h after infection
Sp100 was found predominantly in the typical nuclear dot-like
staining pattern corresponding to ND10s, and there was no
detectable expression of wild-type or mutant IE1-72kDa (Fig.
5a to d and m to p). At 6 h postinfection, both the wild-type
and the IE1-K450R proteins accumulated in the host cell nu-
cleus. Both proteins were found to colocalize with Sp100 in
either nuclear diffuse or punctate patterns, indicating that at
this stage ND10 disruption was complete in some but not all of
the infected cells (Fig. 5e to h and q to t). Finally, at 24 h
postinfection, ND10 disruption was equally complete in all
cells treated with the BADwt or the BADpmIE1-K450R virus,
as the Sp100 protein was displaced from ND10s together with
wild-type IE1-72kDa and IE1-K450R in all infected nuclei,
although some less homogenous Sp100 staining remained (Fig.
5i to l and u to x). Similar results were obtained with infections
performed at a low MOI of 0.01 PFU/cell (data not shown).
These observations clearly show that lack of SUMOylation has
no detectable effect on ND10 disruption by IE1-72kDa.
Besides its interaction with interchromatinic ND10s, IE1-
72kDa is also known to interact with cellular chromatin, at
least during mitosis (2, 32, 70). Since infected cells do not
usually undergo mitosis (10, 35, 36, 69), we analyzed chromatin
association of wild-type and K450R mutant IE1-72kDa by
comparing the localizations of these proteins fused to EGFP in
living transfected cells. As shown in Fig. 4B, both the wild-type
(panel a) and the mutant (panel b) fusion proteins colocalized
very efficiently with condensed chromatin in transfected cells
undergoing mitosis. Taken together, these results show that lack
of SUMOylation has no obvious effects on the subnuclear distri-
bution of IE1-72kDa, including its ability to target to and disrupt
ND10s and to associate with mitotic chromatin. Therefore, it is
unlikely that the observed growth defect of BADpmIE1-K450R
is due to an altered localization of the mutant IE1-72kDa.
Transactivating properties of the mutant proteins. IE1-
72kDa can activate transcription from a variety of HCMV and
other promoters in transient-transfection assays, on its own or
in concert with IE2-86kDa (reviewed in references 13 and 42).
IE1-72kDa is also required for efficient viral early gene expres-
sion in infected cells (20, 22). To investigate whether the
K450R mutation affects the transactivation capacity of IE1-
72kDa, we performed luciferase reporter assays in transiently
transfected cells by using various viral and cellular promoter
constructs. As expected, the wild-type IE1-72kDa protein was
able to activate the SV40 early promoter, the HCMV UL44
promoter, the HCMV major immediate-early promoter, and
the cellular hOrc-1 promoter by factors of 2.6 to 16.1, depend-
ing on the reporter construct (Fig. 6A). IE1-K450R displayed
A
B
FIG. 4. Subcellular localizations and intranuclear interactions of
wild-type and mutant IE1-72kDa proteins in transfected cells. (A) Fi-
broblasts were electroporated with plasmid pCGN-IE1 (panels a to c)
or pCGN-IE1-K450R (panels d to f), and immunofluorescent double-
labeling was performed with mouse anti-PML (5E10) (panels a and d)
and rat anti-HA (3F10) (panels b and e) antibodies, followed by incu-
bation with anti-mouse Alexa 546 (panels a and d) or anti-rat Alexa
488 (panels b and e) conjugates and DNA staining with DAPI (4�,6�-
diamidino-2-phenylindole) (panels c and f). Magnification, �200.
(B) H1299 cells were transfected with plasmids pEGFP-IE1 (panel a)
and pEGFP-IE1-K450R (panel b), and DNA was stained with Hoechst
33342. Living cells that were both successfully transfected and under-
going mitosis were observed by live-cell confocal microscopy. Merged
images are shown, with single-color pictures presented as smaller in-
sets (EGFP, lower left corner; Hoechst 33342, upper left corner).
7808 NEVELS ET AL. J. VIROL.

wild-type-like activity on all three viral promoters, although
transactivation was modestly reduced at the cellular hOrc-1
promoter (1.6- versus 2.8-fold activation) (Fig. 6A). Similar
results were obtained when the same promoters were tested
for synergistic activation by wild-type or mutant IE1-72kDa
and IE2-86kDa, although the SUMOylation-deficient protein
had a tendency to be a slightly more efficient activator in these
assays (Fig. 6B). To confirm these results in the context of viral
replication, we transfected cells with reporter plasmids and
then infected them with the wild-type, K450R, and revertant
viruses. As expected, there were no significant differences in
promoter activation by the three viruses (Fig. 6C). These re-
sults suggest that SUMOylation is not required for transacti-
vation of viral and cellular promoters by IE1-72kDa.
Reduced expression of IE2-86kDa in the IE1-K450R mutant
virus. The fact that we did not find significant differences
between the transactivating properties of the wild-type and the
K450R mutant IE1-72kDa in our reporter assays does not rule
out possible effects on viral gene expression due to differential
regulation of promoters that have not been tested or posttran-
scriptional events. Therefore, we compared the levels of sev-
eral viral transcripts by Northern blotting at various times after
infection of fibroblasts with BADpmK450R-1 or BADrevIE1-1.
The expression kinetics of two immediate-early transcripts,
UL37 and TRS1 (Fig. 7A), as well as the early UL44 mRNA
(not shown), were indistinguishable between the mutant and
revertant viruses. However, the amounts of IE2 mRNA were
substantially reduced in the K450R mutant at all times assayed.
The levels of IE1 mRNA were also reduced, but to a lesser
extent (Fig. 7A). To investigate whether the reduced amount
FIG. 5. ND10 interaction of wild-type and mutant IE1-72kDa
proteins in HCMV-infected cells. Fibroblasts were infected with
BADrevIE1-1 (a to l) or BADpmIE1-K450R-1 (m to x) at a multiplicity
of 3 PFU/cell and fixed 1 h (a to d and m to p), 6 h (e to h and q to t),
or 24 h (i to l and u to x) later. Accumulation of IE1-72kDa proteins
and disruption of ND10s were monitored with antibody combinations
mouse anti-IE1 (1B12)–anti-mouse Alexa 488 (a, e, i, m, q, and u) and
rabbit anti-Sp100–anti-rabbit Alexa 546 (b, f, j, n, r, and v), respec-
tively. DNA was simultaneously stained with DAPI (c, g, k, o, s, and w).
Merged images are also shown (d, h, l, p, t, and x).
FIG. 6. Transcriptional activation by wild-type and mutant IE1-
72kDa proteins. Fibroblasts were transfected with luciferase reporter
plasmid pGL3-Promoter (SV40), pGL3-ICP36 (UL44), pGL3-MIEP
(MIE), or pHsOrc1Luc (Orc-1). Bars represent average values and
standard errors from three separate transfections. Relative light units
are presented as percentages of the respective control value, which was
set to 100%. (A) Respective reporter plasmids were contransfected
with pCGN (black bars) (set to 100%), pCGN-IE1 (gray bars), or
pCGN-IE1-K450R (white bars). (B) Reporter plasmids were cotrans-
fected with pCGN (black bars) (set to 100%) (hardly visible in left
panel), pCGN-IE2 (gray bars), pCGN-IE2 and pCGN-IE1 (white
bars), or pCGN-IE2 and pCGN-IE1-K450R (hatched bars). (C) Trans-
fections with reporter plasmids were followed by infections with
BADwt (gray bars), BADpmIE1-K450R-1 (white bars), or BADrevIE1-1
(hatched bars) or mock infection (black bars) (set to 100%).
VOL. 78, 2004 MUTANT HCMV DEFICIENT FOR SUMOylation OF IE1-72kDa 7809

of IE2 mRNA led to the accumulation of less protein, we
monitored the accumulation of the major IE2 protein species,
IE2-86kDa, by Western blotting in extracts of cells infected
with BADpmIE1-K450R and BADrevIE1 (Fig. 7B). In accor-
dance with the results from our RNA analyses, the accumula-
tion of IE2-86kDa was markedly delayed and reduced in the
mutant, while the levels of IE1-72kDa were, if at all, only
slightly affected. In fact, there was no detectable IE2 protein up
to 24 h after infection with BADpmIE1-K405R at normal film
exposures, compared to readily detectable levels at 16 h postin-
fection with the revertant virus. However, after 48 h the accu-
mulation of IE2-86kDa approached wild-type levels, indicating
that production of the IE2 protein was delayed but not com-
pletely blocked (Fig. 7B). Surprisingly, expression of the UL44
early viral protein was not significantly affected (Fig. 7B), de-
spite the fact that IE2-86kDa has been unequivocally shown to
be required for viral early gene expression (24, 38). This is
most likely due to the fact that the small amounts of IE2 gene
products that are detectable at the RNA (Fig. 7A) but not the
protein (Fig. 7B) level are sufficient to transactivate viral early
promoters. Interestingly, several independent experiments
with different antibodies showed that the SUMOylated form of
IE1-72kDa peaks in abundance at 10 to 18 h postinfection, a
time frame that correlates with the initial accumulation of
IE2-86kDa (Fig. 7B and data not shown). This observation
provides further support for the view that SUMOylation of
IE1-72kDa is linked to efficient expression of the IE2 gene
product. In summary, the growth defect of the IE1-K450R
mutant virus is, at least in part, due to delayed accumulation of
IE2-86kDa.
DISCUSSION
It is well established that IE1-72kDa is required for normal
progression of the HCMV lytic cycle (20, 22, 43), but the exact
role of this protein in viral replication remains elusive. IE1-
72kDa, like the other abundant HCMV major immediate-early
gene product, IE2-86kDa, is a substrate for phosphorylation
(46) and covalent conjugation to the SUMO-1 protein (5, 26,
44, 63, 73) (Fig. 2C and 7B). We have demonstrated that at
least one aspect of its function requires SUMOylation.
Previously generated IE1-deficient viruses are null mutants
that have been constructed by homologous recombination in
mammalian cells followed by plaque purification (22, 43). In
this report we have described the construction and character-
ization of the first IE1 mutant virus generated by homologous
recombination in E. coli with a BAC system, and we have
generated the first virus with point mutations in the IE1-spe-
cific coding sequence. Given the multiplicity-dependent phe-
notype of IE1 null mutant viruses (3, 20, 22, 43), it is not
surprising that HCMV BACs lacking most of the IE1-coding
region (pBADsubIE1) failed to produce virus after transfec-
tion into noncomplementing permissive fibroblasts (Table 1).
However, we were consistently able to reconstitute infectious
virus from pBADpmIE1-K450R, demonstrating that SUMO
modification is not absolutely required for the infectivity of
HCMV BAC DNA and productive viral replication (Table 1).
However, SUMOylation of IE1-72kDa clearly contributes to
the full activity of this protein at both high and low MOIs (Fig.
3). Therefore, it is conceivable that the SUMOylated and non-
conjugated forms of IE1-72kDa may perform distinct roles
during virus infection. The non-SUMOylated protein, which
represents the predominant IE1 species in infected cells (Fig.
2C), may provide the main function that facilitates viral early
gene expression and productive viral replication through
SUMOylation-independent activities such as transcriptional
activation of viral and cellular genes, ND10 disruption, and/or
chromatin binding (20, 22, 43, 63, 73) (Fig. 4 and 5). In con-
trast, the low steady-state levels of SUMOylated IE1-72kDa
evidently have an additional, supporting role that contributes
to efficient lytic viral growth.
Our results indicate that this supporting function is linked to
the induction of IE2 RNA accumulation (Fig. 7). This obser-
vation is somewhat surprising, since a previously characterized
IE1 null virus (CR208) did not exhibit significantly reduced
steady-state levels of IE2-86kDa (3, 20, 22). However, this
recombinant virus was derived from the HCMV Towne strain,
and an earlier mutant (RC303
Acc), which was constructed in
FIG. 7. Comparisons between the temporal pattern of viral RNA
and protein accumulation in cells infected with the K450R mutant and
a revertant virus. (A) Northern blots showing expression of HCMV
IE1, IE2, UL37 exon 1, and TRS1 genes at the indicated times (hours)
after infection (hpi) in fibroblasts infected with the revertant or K450R
mutant virus (3 PFU/cell). As a loading control, 28S and 18S rRNAs
were visualized by staining of the membranes with methylene blue
solution (0.04% in 0.5 M sodium acetate, pH 5.2). (B) Western blot
analysis showing expression of the HCMV IE1-72kDa (SUMOylated
and nonconjugated), IE2-86kDa, and ppUL44 proteins by using anti-
bodies MAB810 (for the IE1 and IE2 proteins) and anti-ICP36 (for
ppUL44), respectively, at the indicated times after infection with wild-
type and K450R mutant viruses (3 PFU/cell). A loading control (�-
tubulin, DM1A) is also included.
7810 NEVELS ET AL. J. VIROL.

a Towne/Toledo hybrid virus background showed substantially
reduced accumulation of IE2-86kDa that was attributed to a
failure in autoregulation (43). Since BADpmIE1-K450R is the
first IE1 mutant virus that is based on HCMV strain AD169, it
is conceivable that interstrain variations contribute to the dif-
ferences in the observed phenotypes. In this context it is inter-
esting that the IE2-86kDa proteins from different HCMV
strains have recently been shown to differ remarkably in vari-
ous biochemical and functional activities (8).
The effect of IE1 SUMOylation on IE2 expression is most
likely a posttranscriptional event rather than a result of tran-
scriptional activation, since both IE1 and IE2 are transcribed
from the major immediate-early promoter and only IE2 ex-
pression is strongly reduced in the K450R mutant virus, while
there is little or no effect on the IE1 RNA and protein levels
(Fig. 7). Moreover, the K450R mutation did not adversely
affect transactivation of the HCMV major immediate-early
promoter by IE1-72kDa in our luciferase reporter assays (Fig.
6). Interestingly, it has been previously reported that normally
discordant expression of the IE1 and IE2 genes at early times
after infection involves posttranscriptional processing events
(64). How SUMOylated IE1-72kDa exerts a posttranscrip-
tional function is presently unknown. However, it is tempting
to speculate that the SUMO moiety may mediate interactions
with components of RNA-processing complexes just like, for
example, SUMOylation of PML mediates the recruitment of
other ND10-targeting proteins such as Sp100, Daxx, and
CREB-binding protein (27, 78). Alternatively, it is conceivable
that SUMOylated IE1-72kDa alters the expression of another
viral or cellular gene product, which then influences the accu-
mulation of IE2 mRNA and protein. Irrespective of its mode
of action, our mutational analysis demonstrates that SUMOy-
lation of IE1-72kDa facilitates viral replication by enhancing
the expression of a second mRNA and protein, IE2-86kDa,
derived from the same transcription unit.
ACKNOWLEDGMENTS
We thank J. Schroeer and J. Goodhouse (Princeton University) for
excellent help with confocal microscopy, R. Greaves (Cambridge Uni-
versity) and E. Mocarski (Stanford University) for the ihf-2 and ih-
fie1.3 cell lines, L. deJong (University of Amsterdam) for antibody
5E10, M. Shirakata (Tokyo Medical and Dental University) for plas-
mid pHsOrc1Luc, C. Paulus (Universitaet Regensburg) for critical
comments on the manuscript and helpful discussions, and Hans Wolf
(Universita¨t Regensburg) for continuous support.
This work was supported by grant CA85786 from the National Can-
cer Institute to T.S. and by Emmy Noether fellowships (NE 791/1-1
and Br1730/2-1) from the Deutsche Forschungsgemeinschaft to M.N.
and W.B, respectively.
REFERENCES
1. Adamson, A. L., and S. Kenney. 2001. Epstein-Barr virus immediate-early
protein BZLF1 is SUMO-1 modified and disrupts promyelocytic leukemia
bodies. J. Virol. 75:2388–2399.
2. Ahn, J. H., E. J. Brignole III, and G. S. Hayward. 1998. Disruption of PML
subnuclear domains by the acidic IE1 protein of human cytomegalovirus is
mediated through interaction with PML and may modulate a RING finger-
dependent cryptic transactivator function of PML. Mol. Cell. Biol. 18:4899–
4913.
3. Ahn, J. H., and G. S. Hayward. 2000. Disruption of PML-associated nuclear
bodies by IE1 correlates with efficient early stages of viral gene expression
and DNA replication in human cytomegalovirus infection. Virology 274:39–
55.
4. Ahn, J. H., and G. S. Hayward. 1997. The major immediate-early proteins
IE1 and IE2 of human cytomegalovirus colocalize with and disrupt PML-
associated nuclear bodies at very early times in infected permissive cells.
J. Virol. 71:4599–4613.
5. Ahn, J. H., Y. Xu, W. J. Jang, M. J. Matunis, and G. S. Hayward. 2001.
Evaluation of interactions of human cytomegalovirus immediate-early IE2
regulatory protein with small ubiquitin-like modifiers and their conjugation
enzyme Ubc9. J. Virol. 75:3859–3872.
6. Atalay, R., A. Zimmermann, M. Wagner, E. Borst, C. Benz, M. Messerle, and
H. Hengel. 2002. Identification and expression of human cytomegalovirus
transcription units coding for two distinct Fc� receptor homologs. J. Virol.
76:8596–8608.
7. Baldick, C. J., Jr., A. Marchini, C. E. Patterson, and T. Shenk. 1997. Human
cytomegalovirus tegument protein pp71 (ppUL82) enhances the infectivity
of viral DNA and accelerates the infectious cycle. J. Virol. 71:4400–4408.
8. Barrasa, M. I., N. Harel, Y. Yu, and J. C. Alwine. 2003. Strain variations in
single amino acids of the 86-kilodalton human cytomegalovirus major im-
mediate-early protein (IE2) affect its functional and biochemical properties:
implications of dynamic protein conformation. J. Virol. 77:4760–4772.
9. Bies, J., J. Markus, and L. Wolff. 2002. Covalent attachment of the SUMO-1
protein to the negative regulatory domain of the c-Myb transcription factor
modifies its stability and transactivation capacity. J. Biol. Chem. 277:8999–
9009.
10. Bresnahan, W. A., I. Boldogh, E. A. Thompson, and T. Albrecht. 1996.
Human cytomegalovirus inhibits cellular DNA synthesis and arrests produc-
tively infected cells in late G1. Virology 224:150–160.
11. Bresnahan, W. A., and T. Shenk. 2000. A subset of viral transcripts packaged
within human cytomegalovirus particles. Science 288:2373–2376.
12. Brune, W., M. Messerle, and U. H. Koszinowski. 2000. Forward with BACs:
new tools for herpesvirus genomics. Trends Genet. 16:254–259.
13. Castillo, J. P., and T. F. Kowalik. 2002. Human cytomegalovirus immediate
early proteins and cell growth control. Gene 290:19–34.
14. Castillo, J. P., A. D. Yurochko, and T. F. Kowalik. 2000. Role of human
cytomegalovirus immediate-early proteins in cell growth control. J. Virol.
74:8028–8037.
15. Cherepanov, P. P., and W. Wackernagel. 1995. Gene disruption in Esche-
richia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision
of the antibiotic-resistance determinant. Gene 158:9–14.
16. Desterro, J. M., M. S. Rodriguez, and R. T. Hay. 1998. SUMO-1 modification
of IkappaBalpha inhibits NF-kappaB activation. Mol. Cell 2:233–239.
17. Duprez, E., A. J. Saurin, J. M. Desterro, V. Lallemand-Breitenbach, K.
Howe, M. N. Boddy, E. Solomon, H. de The, R. T. Hay, and P. S. Freemont.
1999. SUMO-1 modification of the acute promyelocytic leukaemia protein
PML: implications for nuclear localisation. J. Cell Sci. 112:381–393.
18. Endter, C., J. Kzhyshkowska, R. Stauber, and T. Dobner. 2001. SUMO-1
modification required for transformation by adenovirus type 5 early region
1B 55-kDa oncoprotein. Proc. Natl. Acad. Sci. USA 98:11312–11317.
19. Everett, R. D. 2001. DNA viruses and viral proteins that interact with PML
nuclear bodies. Oncogene 20:7266–7273.
20. Gawn, J. M., and R. F. Greaves. 2002. Absence of IE1 p72 protein function
during low-multiplicity infection by human cytomegalovirus results in a
broad block to viral delayed-early gene expression. J. Virol. 76:4441–4455.
21. Gravel, A., J. Gosselin, and L. Flamand. 2002. Human herpesvirus 6 imme-
diate-early 1 protein is a sumoylated nuclear phosphoprotein colocalizing
with promyelocytic leukemia protein-associated nuclear bodies. J. Biol.
Chem. 277:19679–19687.
22. Greaves, R. F., and E. S. Mocarski. 1998. Defective growth correlates with
reduced accumulation of a viral DNA replication protein after low-multi-
plicity infection by a human cytomegalovirus IE1 mutant. J. Virol. 72:366–
379.
23. Hayhurst, G. P., L. A. Bryant, R. C. Caswell, S. M. Walker, and J. H.
Sinclair. 1995. CCAAT box-dependent activation of the TATA-less human
DNA polymerase alpha promoter by the human cytomegalovirus 72-kilodal-
ton major immediate-early protein. J. Virol. 69:182–188.
24. Heider, J. A., W. A. Bresnahan, and T. E. Shenk. 2002. Construction of a
rationally designed human cytomegalovirus variant encoding a temperature-
sensitive immediate-early 2 protein. Proc. Natl. Acad. Sci. USA 99:3141–
3146.
25. Hobom, U., W. Brune, M. Messerle, G. Hahn, and U. H. Koszinowski. 2000.
Fast screening procedures for random transposon libraries of cloned her-
pesvirus genomes: mutational analysis of human cytomegalovirus envelope
glycoprotein genes. J. Virol. 74:7720–7729.
26. Hofmann, H., S. Floss, and T. Stamminger. 2000. Covalent modification of
the transactivator protein IE2-p86 of human cytomegalovirus by conjugation
to the ubiquitin-homologous proteins SUMO-1 and hSMT3b. J. Virol. 74:
2510–2524.
27. Ishov, A. M., A. G. Sotnikov, D. Negorev, O. V. Vladimirova, N. Neff, T.
Kamitani, E. T. Yeh, J. F. Strauss III, and G. G. Maul. 1999. PML is critical
for ND10 formation and recruits the PML-interacting protein daxx to this
nuclear structure when modified by SUMO-1. J. Cell Biol. 147:221–234.
28. Kelly, C., R. Van Driel, and G. W. Wilkinson. 1995. Disruption of PML-
associated nuclear bodies during human cytomegalovirus infection. J. Gen.
Virol. 76:2887–2893.
29. Kim, K. I., S. H. Baek, and C. H. Chung. 2002. Versatile protein tag, SUMO:
its enzymology and biological function. J. Cell Physiol. 191:257–268.
30. Kondo, K., and E. S. Mocarski. 1995. Cytomegalovirus latency and latency-
VOL. 78, 2004 MUTANT HCMV DEFICIENT FOR SUMOylation OF IE1-72kDa 7811

specific transcription in hematopoietic progenitors. Scand. J. Infect. Dis.
Suppl. 99:63–67.
31. Korioth, F., G. G. Maul, B. Plachter, T. Stamminger, and J. Frey. 1996. The
nuclear domain 10 (ND10) is disrupted by the human cytomegalovirus gene
product IE1. Exp. Cell Res. 229:155–158.
32. Lafemina, R. L., M. C. Pizzorno, J. D. Mosca, and G. S. Hayward. 1989.
Expression of the acidic nuclear immediate-early protein (IE1) of human
cytomegalovirus in stable cell lines and its preferential association with
metaphase chromosomes. Virology 172:584–600.
33. Lallemand-Breitenbach, V., J. Zhu, F. Puvion, M. Koken, N. Honore, A.
Doubeikovsky, E. Duprez, P. P. Pandolfi, E. Puvion, P. Freemont, and H. de
The. 2001. Role of promyelocytic leukemia (PML) sumolation in nuclear
body formation, 11S proteasome recruitment, and As2O3-induced PML or
PML/retinoic acid receptor alpha degradation. J. Exp. Med. 193:1361–1371.
34. Lee, E. C., D. Yu, J. Martinez de Velasco, L. Tessarollo, D. A. Swing, D. L.
Court, N. A. Jenkins, and N. G. Copeland. 2001. A highly efficient Esche-
richia coli-based chromosome engineering system adapted for recombino-
genic targeting and subcloning of BAC DNA. Genomics 73:56–65.
35. Lu, M., and T. Shenk. 1996. Human cytomegalovirus infection inhibits cell
cycle progression at multiple points, including the transition from G1 to S.
J. Virol. 70:8850–8857.
36. Lu, M., and T. Shenk. 1999. Human cytomegalovirus UL69 protein induces
cells to accumulate in G1 phase of the cell cycle. J. Virol. 73:676–683.
37. Lukac, D. M., N. Y. Harel, N. Tanese, and J. C. Alwine. 1997. TAF-like
functions of human cytomegalovirus immediate-early proteins. J. Virol. 71:
7227–7239.
38. Marchini, A., H. Liu, and H. Zhu. 2001. Human cytomegalovirus with IE-2
(UL122) deleted fails to express early lytic genes. J. Virol. 75:1870–1878.
39. Margolis, M. J., S. Pajovic, E. L. Wong, M. Wade, R. Jupp, J. A. Nelson, and
J. C. Azizkhan. 1995. Interaction of the 72-kilodalton human cytomegalovi-
rus IE1 gene product with E2F1 coincides with E2F-dependent activation of
dihydrofolate reductase transcription. J. Virol. 69:7759–7767.
40. Messerle, M., G. Hahn, W. Brune, and U. H. Koszinowski. 2000. Cytomeg-
alovirus bacterial artificial chromosomes: a new herpesvirus vector approach.
Adv. Virus Res. 55:463–478.
41. Mitsudomi, T., S. M. Steinberg, M. M. Nau, D. Carbone, D. D’Amico, S.
Bodner, H. K. Oie, R. I. Linnoila, J. L. Mulshine, J. D. Minna, et al. 1992.
p53 gene mutations in non-small-cell lung cancer cell lines and their corre-
lation with the presence of ras mutations and clinical features. Oncogene
7:171–180.
42. Mocarski, E. S., and C. T. Courcelle. 2001. Cytomegaloviruses and their
replication, p. 2629–2673. In D. M. Knipe, P. M. Howley, et al. (ed.), Fields
virology, 4th ed., vol. 2. Lippincott Williams and Wilkins, Philadelphia, Pa.
43. Mocarski, E. S., G. W. Kemble, J. M. Lyle, and R. F. Greaves. 1996. A
deletion mutant in the human cytomegalovirus gene encoding IE1(491aa) is
replication defective due to a failure in autoregulation. Proc. Natl. Acad. Sci.
USA 93:11321–11326.
44. Muller, S., and A. Dejean. 1999. Viral immediate-early proteins abrogate the
modification by SUMO-1 of PML and Sp100 proteins, correlating with
nuclear body disruption. J. Virol. 73:5137–5143.
45. Muller, S., M. J. Matunis, and A. Dejean. 1998. Conjugation with the ubiq-
uitin-related modifier SUMO-1 regulates the partitioning of PML within the
nucleus. EMBO J. 17:61–70.
46. Pajovic, S., E. L. Wong, A. R. Black, and J. C. Azizkhan. 1997. Identification
of a viral kinase that phosphorylates specific E2Fs and pocket proteins. Mol.
Cell. Biol. 17:6459–6464.
47. Pari, G. S., and D. G. Anders. 1993. Eleven loci encoding trans-acting factors
are required for transient complementation of human cytomegalovirus ori-
Lyt-dependent DNA replication. J. Virol. 67:6979–6988.
48. Pichler, A., and F. Melchior. 2002. Ubiquitin-related modifier SUMO1 and
nucleocytoplasmic transport. Traffic 3:381–387.
49. Poma, E. E., T. F. Kowalik, L. Zhu, J. H. Sinclair, and E. S. Huang. 1996.
The human cytomegalovirus IE1–72 protein interacts with the cellular p107
protein and relieves p107-mediated transcriptional repression of an E2F-
responsive promoter. J. Virol. 70:7867–7877.
50. Posfai, G., M. D. Koob, H. A. Kirkpatrick, and F. R. Blattner. 1997. Versatile
insertion plasmids for targeted genome manipulations in bacteria: isolation,
deletion, and rescue of the pathogenicity island LEE of the Escherichia coli
O157:H7 genome. J. Bacteriol. 179:4426–4428.
51. Rangasamy, D., and V. G. Wilson. 2000. Bovine papillomavirus E1 protein is
sumoylated by the host cell Ubc9 protein. J. Biol. Chem. 275:30487–30495.
52. Rangasamy, D., K. Woytek, S. A. Khan, and V. G. Wilson. 2000. SUMO-1
modification of bovine papillomavirus E1 protein is required for intranuclear
accumulation. J. Biol. Chem. 275:37999–38004.
53. Reddehase, M. J. 2000. The immunogenicity of human and murine cyto-
megaloviruses. Curr. Opin. Immunol. 12:390–396.
54. Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty per
cent endpoints. Am. J. Hyg. 27:493–497.
55. Regad, T., and M. K. Chelbi-Alix. 2001. Role and fate of PML nuclear bodies
in response to interferon and viral infections. Oncogene 20:7274–7286.
56. Rogan, S., and S. Heaphy. 2000. The vaccinia virus E3L protein interacts
with SUMO-1 and ribosomal protein L23a in a yeast two hybrid assay. Virus
Genes 21:193–195.
57. Romanowski, M. J., and T. Shenk. 1997. Characterization of the human
cytomegalovirus irs1 and trs1 genes: a second immediate-early transcription
unit within irs1 whose product antagonizes transcriptional activation. J. Vi-
rol. 71:1485–1496.
58. Sarisky, R. T., and G. S. Hayward. 1996. Evidence that the UL84 gene
product of human cytomegalovirus is essential for promoting oriLyt-depen-
dent DNA replication and formation of replication compartments in cotrans-
fection assays. J. Virol. 70:7398–7413.
59. Schwartz, D. C., and M. Hochstrasser. 2003. A superfamily of protein tags:
ubiquitin, SUMO and related modifiers. Trends Biochem. Sci. 28:321–328.
60. Seeler, J. S., and A. Dejean. 2003. Nuclear and unclear functions of SUMO.
Nat. Rev. Mol. Cell. Biol. 4:690–699.
61. Shen, Y., H. Zhu, and T. Shenk. 1997. Human cytomegalovirus IE1 and IE2
proteins are mutagenic and mediate “hit-and-run” oncogenic transformation
in cooperation with the adenovirus E1A proteins. Proc. Natl. Acad. Sci. USA
94:3341–3345.
62. Shirakata, M., M. Terauchi, M. Ablikim, K. Imadome, K. Hirai, T. Aso, and
Y. Yamanashi. 2002. Novel immediate-early protein IE19 of human cyto-
megalovirus activates the origin recognition complex I promoter in a coop-
erative manner with IE72. J. Virol. 76:3158–3167.
63. Spengler, M. L., K. Kurapatwinski, A. R. Black, and J. Azizkhan-Clifford.
2002. SUMO-1 modification of human cytomegalovirus IE1/IE72. J. Virol.
76:2990–2996.
64. Stamminger, T., E. Puchtler, and B. Fleckenstein. 1991. Discordant expres-
sion of the immediate-early 1 and 2 gene regions of human cytomegalovirus
at early times after infection involves posttranscriptional processing events.
J. Virol. 65:2273–2282.
65. Stuurman, N., A. de Graaf, A. Floore, A. Josso, B. Humbel, L. de Jong, and
R. van Driel. 1992. A monoclonal antibody recognizing nuclear matrix-
associated nuclear bodies. J. Cell Sci. 101:773–784.
66. Szostecki, C., H. H. Guldner, H. J. Netter, and H. Will. 1990. Isolation and
characterization of cDNA encoding a human nuclear antigen predominantly
recognized by autoantibodies from patients with primary biliary cirrhosis.
J. Immunol. 145:4338–4347.
67. Verger, A., J. Perdomo, and M. Crossley. 2003. Modification with SUMO. A
role in transcriptional regulation. EMBO Rep. 4:137–142.
68. Wagner, M., Ruzsics, Z., Koszinowski, U. H. 2002. Herpesvirus genetics has
come of age. Trends Microbiol. 10:318–324.
69. Wiebusch, L., and C. Hagemeier. 1999. Human cytomegalovirus 86-kilodal-
ton IE2 protein blocks cell cycle progression in G1. J. Virol. 73:9274–9283.
70. Wilkinson, G. W., C. Kelly, J. H. Sinclair, and C. Rickards. 1998. Disruption
of PML-associated nuclear bodies mediated by the human cytomegalovirus
major immediate early gene product. J. Gen. Virol. 79:1233–1245.
71. Wilson, V. G., and D. Rangasamy. 2001. Intracellular targeting of proteins by
sumoylation. Exp. Cell Res. 271:57–65.
72. Wilson, V. G., and D. Rangasamy. 2001. Viral interaction with the host cell
sumoylation system. Virus Res. 81:17–27.
73. Xu, Y., J. H. Ahn, M. Cheng, C. M. apRhys, C. J. Chiou, J. Zong, M. J.
Matunis, and G. S. Hayward. 2001. 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. J. Virol. 75:10683–
10695.
74. Yu, D., H. M. Ellis, E. C. Lee, N. A. Jenkins, N. G. Copeland, and D. L.
Court. 2000. An efficient recombination system for chromosome engineering
in Escherichia coli. Proc. Natl. Acad. Sci. USA 97:5978–5983.
75. Yu, Y., and J. C. Alwine. 2002. Human cytomegalovirus major immediate-
early proteins and simian virus 40 large T antigen can inhibit apoptosis
through activation of the phosphatidylinositide 3�-OH kinase pathway and
the cellular kinase Akt. J. Virol. 76:3731–3738.
76. Yurochko, A. D., M. W. Mayo, E. E. Poma, A. S. Baldwin, Jr., and E. S.
Huang. 1997. Induction of the transcription factor Sp1 during human cyto-
megalovirus infection mediates upregulation of the p65 and p105/p50 NF-�B
promoters. J. Virol. 71:4638–4648.
77. Zhang, Y., F. Buchholz, J. P. Muyrers, and A. F. Stewart. 1998. A new logic
for DNA engineering using recombination in Escherichia coli. Nat. Genet.
20:123–128.
78. Zhong, S., S. Muller, S. Ronchetti, P. S. Freemont, A. Dejean, and P. P.
Pandolfi. 2000. Role of SUMO-1-modified PML in nuclear body formation.
Blood 95:2748–2752.
79. Zhu, H., Y. Shen, and T. Shenk. 1995. Human cytomegalovirus IE1 and IE2
proteins block apoptosis. J. Virol. 69:7960–7970.
7812 NEVELS ET AL. J. VIROL.