1. Art and Diseño

JOURNAL OF VIROLOGY, Nov. 1994, p. 7549-7553
Vol. 68, No. 11
0022-538X/94/$04.00+0
Copyright X 1994, American Society for Microbiology
Protein-Protein Interactions between Human Cytomegalovirus
IE2-580aa and pUL84 in Lytically Infected Cells
DAVID J. SPECTOR* AND MARY J. TEVETHIA
Department of Microbiology and Immunology, Program in Cell and Molecular Biology, and Intercollege Program in
Genetics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
'Received 25 May 1994/Accepted 3 August 1994
Only the broad outlines of the lytic cycle of human cytomegalovirus (HCMV) are known. Like those of other DNA
viruses, HCMV gene products exhibit cascade regulation, with
the onset of viral DNA replication providing a separation
between prereplicative (early) and postreplicative (late) expression. Limited viral gene expression, further demarked as
immediate early, occurs in the absence of viral protein synthesis (7, 8, 24, 43, 44). However, only a handful of viral functions
are implicated in regulating this cascade. Among these is the
580-amino-acid (in strain AD169 [3]) product of the immediate-early region 2 gene (IE2-580aa) (19).
IE2-580aa is encoded by three exons, and a fourth, noncoding exon specifies a 5' untranslated region (see Fig. 1) (23, 31,
38). It is a nuclear phosphoprotein (15, 31) that activates
transcription of HCMV genes (2, 23, 39), as well as heterologous viral and cellular genes (6, 10, 12, 15, 32, 34, 41). The
protein also represses its own transcription (14, 32) and that of
the 491-amino-acid immediate-early region 1 protein (IE1491aa) (40), with which IE2-580aa shares 85 amino-terminal
residues (see Fig. 1). Repression is accomplished by binding to
a specific sequence overlapping the initiation site for transcription of the common precursor to the IE1 and IE2 mRNAs (4,
5, 14, 16, 20-22, 30). The capacity of IE2-580aa to activate
transcription of other genes and repress its own implicates this
protein as a central player in regulation of the HCMV lytic
cycle, as well as alternative life cycles, such as latency or
persistence.
Like the products of the other immediate-early genes of
DNA viruses, IE2-580aa binds to both cellular and viral
proteins. In vitro interactions with a number of cellular proteins have been reported (9). Those that occur with the general
transcription factors TATA-binding protein and TFIIB link
IE2-580aa with core components of the transcription apparatus (1, 12, 17). In addition, IE2-580aa self-associates (5, 9),
although the role of this interaction is not known.
We recently reported that antibodies to IE2-580aa coprecipitate a 75-kDa protein (p75) from lysates of productively
infected human embryonic lung (HEL) cells (36). On the basis
of its altered electrophoretic mobility when precipitated from
cells infected with two different viral strains, AD169 and
Towne, we predicted that p75 is of viral origin. Identification of
the p75 gene is a prerequisite to investigating the consequences
of this interaction for both proteins. Here we show that p75 is
the product of the UL84 gene (pUL84; Fig. 1) (13), which has
been implicated in replication of HCMV DNA (29).
For these experiments, mycoplasma-free cultures of HEL
cells were maintained in monolayer culture (36). HCMV
strains AD169 (ATCC VR-538) and Towne (ATCC VR-977)
were obtained from the American Type Culture Collection,
and stocks were prepared in HEL cells and assayed by plaque
titration (36). 35S-labeled polypeptides isolated from infected
cells were analyzed by immunoprecipitation and immunoblotting as described previously (36). Electrophoresis was done
with 7.5% polyacrylamide gels containing an acrylamide-bisacrylamide ratio of 30:0.5 at 0.54 mA/cm for 20 to 22 h. Table 1
lists the properties of the antibodies used. Antiserum 901 was
provided by S. S. Tevethia. Antibody NEA 9221 was purchased
from Dupont NEN Research Products. Antibodies 2183 and
1218 were provided by J. Nelson. Antibody CH41 was obtained
from L. Pereira. Antibody to a UL84-glutathione S-transferase
(GST) fusion protein was provided by E.-S. Huang.
HCMV proteins with molecular sizes and properties similar
to those of p75 were considered as candidates for the unknown
polypeptide. Among these are pUL84, the product of the
UL84 gene (13), and infected-cell protein 22 (ICP22), the
US22 gene product (25). We obtained antiserum to a UL84GST fusion protein (anti-UL84 serum [13]), as well as a
monoclonal antibody (CH41) to ICP22 (25). When 35S-labeled, HCMV-infected cell lysates prepared at 24 h postinfection (hpi) were immunoprecipitated with antiserum to IE2580aa (1218) or the UL84 protein, similar polypeptide profiles
were observed (Fig. 2A). Antibody 1218 precipitated the
mature 86-kDa form of IE2-580aa (the immature 80-kDa form
is not very apparent in cells labeled for 2 h [36]) and the
coprecipitating protein, p75 (36), as well as less abundant
species. As reported previously, the IE2-580aa and p75 proteins from strain AD169- and Towne-infected cells had different electrophoretic mobilities. Bands with corresponding mobilities also were precipitated by anti-UL84 serum. In addition,
there was a band migrating slightly faster than, and with the
same strain-dependent mobility difference as, the respective
p75-like species.
A more extensive panel of antibodies was reacted with
labeled extracts prepared at 48 hpi (Fig. 2B). Once again, the
IE2 protein and p75 profiles obtained with antibodies to IE2
*
Corresponding author. Phone: (717) 531-8250. Fax: (717) 5316522. Electronic mail address: [email protected].
7549
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The human cytomegalovirus immediate-early protein IE2-580aa (ppUL122a) activates transcription of viral
and cellular genes and represses its own transcription through sequence-specific binding to the major
immediate-early promoter. In lytically infected cells, IE2-580aa interacts with a 75-kDa viral protein (p75), an
early protein that is also synthesized at late times after infection. Here we show that p75 is the product of the
UL84 gene. Its association with IE2-580aa in infected cells suggests that pUL84 is involved in transcription
control.
7550
NOTES
J. VIROL.
A
0.0
I
0.1
0.2
0.3
0.4
0.5
I
I
I
I
I
0.6
0.7
I
I
0.8
I
B
C
B
0.54
0.525
UL84
121312
123.00
c
0.735
UL122
ATG
UL123
S5 26
60a 401
56
L
160*57
1708S1 171.000
172225
(1218) were reproduced with anti-UL84 serum. The relative
amount of IE2 protein recovered with anti-UL84 serum was
reduced compared with the 24-h sample. Again, the band
migrating slightly faster than p75 was observed with the
anti-UL84 serum. The identity of this band was not determined; it may be a proteolytic product. In addition, this serum
also precipitated a 90-kDa protein with slightly faster mobility
from strain Towne-infected cells. This protein probably corresponds to the 90-kDa UL84-related protein observed at late
times by He et al. (13). Abundant 63-kDa (p63) and 40-kDa
proteins also were detected with the two antisera. These data
suggest that p75 is pUL84 and that the UL84 antiserum
coprecipitated the IE2 protein.
Antibody CH41 precipitated a protein of about 70 kDa (as
expected for ICP22) that was clearly different from p75. As
expected, antibody 2183 precipitated IE1-491aa (the AD169
protein migrated faster than the Towne counterpart [36]),
TABLE 1. Antibodies and their specificities
Designation
9221
Typea
Protein(s)
recognized
MAb IE1-491aa
Amino acid
positions
defining epitope
1-24
Reference(s)
383-420
548-564
26, 38
15
13
25
42
33
IE2-580aa
2183
1218
UL84
CH41
901
Pep
Pep
GST
MAb
MAb
IE1-491aa
IE2-580aa
pUL84
pUS22
SV40C large T
NDb
684-698
aPep, rabbit antipeptide serum; MAb, mouse monoclonal antibody; GST,
rabbit antiserum to GST fusion protein.
bND, not determined.
SV40, simian virus 40.
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FIG. 1. Map of the HCMV genome with expansion of the regions
encoding pUL84 and IE2-580aa (3, 13, 31, 38, 40). The locations of the
expanded regions (B and C) on the linear HCMV genome map (strain
AD169; 229,354 bp; 0.01 map unit = 2,294 bp) are shown in panel A.
Panel B shows the location of the open reading frame encoding
pUL84. Panel C shows the location of the open reading frames
encoding IE2-580aa and IE1-491aa. For simplicity, other IE1 and IE2
products are not shown. The exon structures for the mRNAs are
indicated below the line. For panels B and C, the arrows show the
direction of transcription. The numbers above the reading frames
indicate the amino acid residues in each protein encoded by each
segment (the first coding exon for IEl and IE2 encodes amino acids 1
to 24 of each). The nucleotide positions of the beginning and end of
each long reading frame are shown immediately below the diagrams.
whereas antibody 9221, which recognizes the common region
of IEl and IE2 proteins, produced profiles similar to those
obtained with antibody 1218. The lot of 9221 antibody used in
these experiments did not recognize IE1-491aa as well as some
others. Unexpectedly, all of the antibodies, including the
simian virus 40 large-T-antigen-specific monoclonal antibody
(901), precipitated the 63-kDa species to some degree.
The UL84 gene product has a predicted molecular mass of
65.4 kDa (3, 13). The precise electrophoretic mobility of a
protein relative to that of standards does not always agree with
the prediction. Nevertheless, p75 could not be assigned to
UL84 solely on the basis of the data in Fig. 2. To determine
which immunoprecipitated species is pUL84, the electrophoretically separated proteins labeled with [35S]methionine
were blotted to nitrocellulose (Fig. 3). An autoradiogram of
the blot (left panel) revealed the three abundant proteins,
IE2-580aa, p75, and p63, immunoprecipitated by antiserum
against IE2 (1218). The same three labeled proteins were
immunoprecipitated by antiserum against the shared IE1-IE2
epitope (9221), although as in Fig. 2B, the amount of p63 was
reduced. Antibody against TEl (2183) precipitated IE1-491aa
and a band similar in mobility to p63. The antibody to the
simian virus 40 large T antigen did not react with any ICPs.
When the blots were probed with anti-UL84 serum (right
panel), the lanes containing labeled p75 revealed an immunoreactive band with the same mobility.
To confirm the assignment of p75 to the UL84 gene, the
different mobilities of the p75 bands in HCMV strain Towneand AD169-infected cells were exploited. Labeled proteins
immunoprecipitated by antibody 1218 from HEL cells infected
with either virus were blotted. Immunoprecipitation of AD169infected cell extracts with the monoclonal antibody (CH41) to
ICP22 provided a specificity control. The autoradiogram of the
blot (Fig. 4, left panel) shows that, as expected, Towne p75
migrated slightly faster than the AD169 counterpart. The
opposite was true for the IE2 protein, and the two p63 species
migrated identically. Antibody CH41 precipitated 70-kDa protein ICP22, as well as a band with the same mobility as p63.
When the blot was probed with anti-UL84 serum, the direct
immunological detection revealed that pUL84 had the same
strain-dependent migration as p75. We concluded that p75 and
pUL84 are identical.
There also was a faint background band in the region of p63
in all lanes of the probed blot in Fig. 4. The apparent ubiquity
of p63 in both immunoprecipitation and blot experiments
raised obvious questions as to its identity. We reported previously the detection of a band with similar mobility, although it
did not appear to be as abundant as in these experiments (36).
On the basis of its antibody reactivity profile, we suggested that
it was IE2-425aa, a protein made from an IE2 mRNA with an
exon structure different from that encoding IE2-580aa (31, 38).
However, monoclonal antibodies (18) against tegument protein LMP (11, 35), the product of the UL83 gene (3, 27, 28),
reacted strongly with blotted p63 (37). Therefore, p63 probably
is LMP, a very abundant protein in late-infected cells. We
suspect that p63 was observed more easily than before in the
experiments shown in Fig. 2 to 4, especially at 48 hpi, because
of more synchronous infection conditions. We achieved multiplicities of infection that were consistently higher than those
used previously. The results probably reflect, at least in part,
the nonspecific association of p63 with immunoprecipitates
and nonspecific detection in blots, although specific binding
has not been ruled out. Further investigation is required to
determine if p63 binds specifically to other viral polypeptides.
The specificity of the IE2-580aa-pUL84 interaction, on the
other hand, has been established (36). Relatively little is known
VOL. 68, 1994
C.
NOTES
1218
UL84
A T Mo A T
A
A T A T
A T A T A T
M
A T
-97.4
-97.4
ICC{~~~~~~~~~~~~~
A.
_
UL84 2183 CH41 901
B 9221 1218
M
7551
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-68.0
*
,i," t
_a
IE2-c
p75
.41s
4W0
*,a
__
:
s|
~p63
_
-43.0
-68.0
_
il
q"o
i.
K
`::..''A
I.Wf-
-
43.0
about pUL84, an early protein that continues to be made at
late times after infection but has not been found in virions (13).
Provocatively, there is a leucine heptameric repeat region
between amino acids 114 and 135 that could participate in
protein-protein interactions. There is also an octameric leucine
repeat between amino acids 325 and 349. The UL84 gene is
implicated in HCMV DNA replication: UL84 is among 11
genetic loci, including TEl and IE2, required for complementation of HCMV oriLyt-dependent DNA replication in a
transient transfection system (29). pUL84 could be a replication protein. If so, the interaction with IE2-580aa could recruit
the immediate-early protein to the replication complex, where
it might function in transcriptional activation of the initiation
of viral DNA synthesis. Alternatively, the interaction between
the pUL84 protein and IE2-580aa might modify the target
specificity of the transcriptional regulatory activity of either
protein so that promoters of DNA replication genes are
activated.
Association with IE2-580aa could be the normal state for
intracellular pUL84. Since the amounts of pUL84 in antiUL84 immune complexes did not differ substantially from the
amounts in immune complexes formed with IE2-specific antibodies, there may not be a large pool of free pUL84 in the cell.
We obtained no information as to whether the complexes
AD To AD
AD To AD
a)
a)
19t co
X
Orcs
-
I:
Eo
CO
CO
Y
rl,
I-,l
c
97.4
97.4
-
-
o
68.0
-
_
_
-
--
I1E2
p75
p63
_tso
n-
IE2c
----=-
p75 -a
68.0
-
owl
-
p3-
'.-
blot probed with
anti-UL84 serum
FIG. 3. Identification of pUL84 in immunoprecipitated samples.
HEL cells infected with HCMV strain AD169 at a multiplicity of
infection of 3 were labeled with [35S]methionine at 44 to 46 hpi and
chased for 1 h prior to sample preparation. Cell extracts were
immunoprecipitated with the antibodies indicated above the lanes.
After electrophoretic separation of the immunoprecipitated, radiolabeled proteins, they were blotted to nitrocellulose, probed with
anti-UL84 serum (right panel), and exposed to film for autoradiography (left panel) as described previously (36). The positions of IE2580aa (IE2), p75, and p63 are indicated between the panels. The sizes
(in kilodaltons) of molecular mass standards are shown on the left.
blot probed with
anti-UL84 serum
FIG. 4. Comparison of the HCMV strain-dependent mobilities of
p75 and pUL84. HEL cells were infected with HCMV strain AD169
(AD; multiplicity of infection of 3) or Towne (To; multiplicity of
infection of 2), labeled with [35S]methionine at 73 to 75 hpi, and chased
for 1 h prior to sample preparation. Cell extracts were immunoprecipitated with the antibodies indicated and processed for immunoblotting. The blots were probed with anti-UL84 serum (right panel) and
exposed to film for autoradiography (left panel). The positions of
IE2-580aa (IE2), p75, and p63 are indicated between the panels. The
sizes (in kilodaltons) of molecular mass standards are shown on the
left.
Downloaded from http://jvi.asm.org/ on February 6, 2015 by guest
'n."
FIG. 2. Comparison of ICPs immunoprecipitated by antisera to HCMV proteins. HEL cells infected with HCMV strain AD169 (A) at a
multiplicity of infection of 3 or with strain Towne (T) at a multiplicity of infection of 2 or mock infected (Mo) were labeled with [35S]methionine
for 2 h. Cell extract proteins were immunoprecipitated with the antibodies indicated. (A) Samples prepared at 24 hpi. The positions of IE2-580aa
(IE2) and p75 are indicated on the left. The sizes of the molecular mass standards in lane M are shown in kilodaltons on the right. (B) Samples
prepared at 48 hpi. The positions of IE2-580aa (IE2), p75, and p63 are indicated between the panels. The sizes (in kilodaltons) of the molecular
mass standards in lane M are shown on the right.
7552
J. VIROL.
NOTES
containing IE2-580aa and pUL84 also include other proteins.
Others have observed multiple interactions with IE2-580aa in
vitro (9). If these data reflect in vivo interactions, at least some
of the protein complexes also might contain pUL84.
IE2-580aa contains two nuclear localization signals (31),
whereas no such signals have been identified in pUL84, which
changes its subcellular localization from perinuclear to mostly
cytoplasmic to mostly nuclear as infection progresses (13). The
interaction of these proteins could allow a signal in one protein
to target the other to the nucleus. On the other hand, binding
could obscure a targeting signal in one of the proteins,
resulting in altered compartmentalization of the complexed
form.
J. Virol. 66:95-105.
7. DeMarchi, J. M. 1981. Human cytomegalovirus DNA: restriction
enzyme cleavage maps and map locations for immediate early,
early and late RNAs. Virology 114:23-38.
8. DeMarchi, J. M., C. A. Schmidt, and A. S. Kaplan. 1980. Pattern
of transcription of human cytomegalovirus in permissively infected
cells. J. Virol. 35:277-286.
9. Furnari, B. A., E. Poma, T. F. Kowalik, S.-M. Huong, and E.-S.
Huang. 1993. Human cytomegalovirus immediate-early gene 2
protein interacts with itself and with several novel cellular proteins. J. Virol. 67:4981-4991.
10. Ghazal, P., J. Young, E. Giulietti, C. DeMattei, J. Garcia, R
Gaynor, R. M. Stenberg, and J. A. Nelson. 1991. A discrete cis
element in the human immunodeficiency virus long terminal
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cytomegaloviruses. Virology 128:391-406.
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14.
15.
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We thank the following investigators, who graciously and generously
provided antibodies for these experiments: E.-S. Huang, G. LaFauci, J.
Nelson, L. Pereira, and S. S. Tevethia. John Wills and Lorna Samaniego provided valuable comments on the manuscript. We also
thank Tim Grierson for photography.
This work was supported by Public Health Service program project
grant CA27503 from the National Cancer Institute and by a grant from
the Biomedical Research Support Grant Program, National Institutes
of Health (RR05680).
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NOTES