Previous Article | Next Article 
Journal of Virology, July 1999, p. 6073-6075, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Novel Structural Protein of Human
Cytomegalovirus, pUL25, Is Localized in the Viral Tegument
N.
Zini,1
M.
C.
Battista,2
S.
Santi,1
M.
Riccio,1
G.
Bergamini,2
M. P.
Landini,2,* and
N. M.
Maraldi1
Institute of Normal and Pathologic
Cytomorphology, C.N.R., c/o I.O.R.,1 and
Department of Clinical and Experimental Medicine, Division of
Microbiology, University of Bologna, St. Orsola
Hospital,2 Bologna, Italy
Received 8 February 1999/Accepted 19 March 1999
 |
ABSTRACT |
Human cytomegalovirus UL25 codes for a structural phosphoprotein of
85 kDa (C. J. Baldick and T. Shenk, J. Virol. 70:6097-6105, 1996; M. C. Battista et al., J. Virol. 73:3800-3809, 1999).
In this study we analyzed the intracellular and intraviral localization of pUL25 by confocal and immunoelectron microscopy and found that pUL25
is a component of the viral tegument and the dense body matrix,
acquired during the late cytoplasmic phase of virus maturation.
| |
TEXT |
The human cytomegalovirus (CMV)
virion consists of an icosahedral capsid embedded in an amorphous
layer, called the tegument, that fills the space between the capsid and
the trilaminar external envelope. The tegument contains approximately
40% of the virion protein mass (7), but little is known
about its structure or function. Its protein composition remains
incompletely defined, even though the following gene products have been
assigned to it: pUL48, pUL47, pUL32, pUL82, pUL83, and pUL99. Other
possible tegument components are a subform of pUL69, pUL56, pUL94, and pUL97 (for a review, see references 5 and
14).
CMV UL25 has recently been found to encode a new structural protein,
present in both virions and defective virus particles (1).
In a more-recent work we defined the main characteristics of pUL25, a
phosphoprotein of 85 kDa expressed with true late kinetics
(2). Because of its presence both in virions and in the
dense bodies and because of its intracellular colocalization with other
tegument proteins, such as pUL99, it has been suggested that pUL25 is a
new component of the viral tegument (1, 2). In this study
confocal microscopy and immunoelectron microscopy were used to define
the intracellular and intraviral localization of pUL25. Human embryonic
lung (HEL) fibroblasts were grown in Eagle's minimum essential medium
supplemented with 10% fetal calf serum. Infections were made on 70 to
80% confluent monolayers of HEL cells with the CMV strain AD169 at a
multiplicity of infection of 1 to 2 for 60 min.
pUL25 expression was determined by indirect immunofluorescence, after
fixation with 4% paraformaldehyde for 15 min at 4°C, on CMV-infected
glass-adherent HEL cells incubated with polyclonal antibody CK25 in
ascitic fluid (2) (dilution, 1:400) for 1 h at 37°C
in a humid chamber. A fluorescein-conjugated goat anti-mouse Tec-Fab2 GAM-Ig (Technogenetics, Milan, Italy) was used as
a secondary antibody. Fluorescent samples were imaged with a Phoibos
1000 confocal system (Molecular Dynamics, Sunnyvale, Calif.). For
three-dimensional reconstruction, 0.3-µm-thick serial optical
sections were recorded (512 by 512 pixels) and processed by dedicated
software (ImageSpace; Molecular Dynamics) running on an Indigo
workstation (Silicon Graphics, Mountain View, Calif.). pUL25
subcellular localization was analyzed by pseudocolor representation of
fluorescence intensity on phase-contrast microscopy-imaged cells.
For the immunocytochemical detection of pUL25, the pelleted cells were
fixed with 1% glutaraldehyde for 30 min, dehydrated with absolute
ethanol, and embedded in Epon. Thin sections were treated for 10 min
with 10% H2O2, preincubated with 5% normal goat serum, and incubated with polyclonal antibody CK25 in ascitic fluid (dilution, 1:100). A goat anti-mouse immunoglobulin G conjugated with 10-nm-diameter colloidal gold particles was used as a secondary antibody. Silver enhancement (Silver Enhancer Kit; Amersham, Little Chalfont, Buckinghamshire, United Kingdom) was used to enlarge the gold
particle size. Controls consisted of samples not included with the
primary antibody. As shown in Fig. 1A, a
very low-level background signal was detected in mock-infected HEL
cells. In infected HEL cells, starting from 48 h postinfection
(p.i.) and increasing thereafter, a strong signal, scattered within the
cytoplasm and always localized in that cell compartment, was obtained
(Fig. 1B). The signal showed a grainy consistency, similar to that
described for the pUL25 murine homologue (4).
View larger version (151K):
[in this window]
[in a new window]
|
FIG. 1.
(A and B) Confocal microscopy of HEL cells labeled with
anti-pUL25 antibody. (A) Mock-infected cells show a very faint
background signal. (B) At 96 h after infection with CMV, the
fluorescence due to the anti-pUL25 antibody in the cytoplasm indicates
the presence of the protein in cytoplasmic bodies of different sizes.
Bar = 5 µm. (C through G) Electron microscopy of HEL cells
stained with anti-pUL25 antibody 120 h after infection. (C) Gold
particles are present on CDB and on virion particles generally devoid
of a core. The majority of the gold particles are localized along the
tegument (arrows). (D and E) High magnification of intracytoplasmic
virions in which the core is visible. The labeling is less intense on
the tegument than in the virions from which the core has been removed
by the section etching, and the tegument is more available to antibody
binding (panel C). (F) Extracellular enveloped virions are labeled on
the tegument (arrow). An immunolabeled dense body is also present in
the extracellular space (arrowhead). (G) Detail of an extracellular
virion which is tangentially sectioned, thus not allowing the detection
of the core, which presents intense labeling on the tegument. Bar = 0.1 µm.
|
|
The results obtained indicate that pUL25 is expressed exclusively at
the cytoplasmic level during the late phase of the virus replication
cycle, as previously reported for other human CMV tegument proteins,
such as pUL99 (10).
In agreement with the data obtained by confocal microscopy, electron
microscopy immunolabeling studies revealed completely negative results
until 48 h p.i. Thereafter, typical cytoplasmic structures, unique
to CMV and called cytoplasmic dense bodies (CDB [17]),
were increasingly positive with the anti-pUL25 antibody (Fig. 1C). The
HEL cells, at 5 days p.i., presented virions at the different
maturation stages, both in the nucleus and in the cytoplasm. The gold
labeling due to the anti-pUL25 antibody was present exclusively on
cytoplasmic virions, generally localized along the inner surface facing
either the core or the empty space in which the core was no longer
present (probably because of the "etching effect") (Fig. 1C through
E). Extracellular virus particles showed a labeling pattern identical
to that observed on the intracellular particles (Fig. 1F and G). No
labeling was detected on the nucleocapsids or on the envelopes of
intracellular or extracellular virions (data not shown). Therefore, the
presence of the bulk of the observed labeling distribution within the
virion particles suggests that CMV pUL25, like its human herpesvirus 7 counterpart U14 (15), is present in the viral tegument
during cytoplasmic virion assembly as well as in mature extracellular virions.
Our findings strongly suggest that in addition to tegument proteins,
such as pUL82, pUL83, pUL32, and pUL69, which are translocated into the
nucleus and probably associate with the nucleocapsid within this cell
compartment (6, 8, 9, 11, 12, 17), there are other tegument
proteins, such as pUL99 and pUL25, which do not enter the nucleus and
associate with the nucleocapsid in the cytoplasm, where envelopment
also occurs (8, 13, 16). Envelopment is most likely mediated
by a specific recognition between a viral protein(s) residing within
the tegument and a viral protein(s) present in the cytoplasmic
membranes. We suggest that the tegument proteins which are expressed
exclusively in the cytoplasm and during the late phases of virus
replication are the most suitable for envelopment, being probably the
last to be incorporated. pUL25 seems to be a new candidate for this role.
| |
ACKNOWLEDGMENTS |
We thank Luisa Bertacchi for excellent technical assistance and
Aurelio Valmori for photographic work.
This work was supported by the Italian Ministry of University and
Scientific Research and by the University of Bologna (60 and 40%); the
work was also supported by the AIDS Projects of the Italian Ministry of
Public Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Clinical and Experimental Medicine, Division of Microbiology,
University of Bologna, St. Orsola General Hospital, Via Massarenti 9, 40138 Bologna, Italy. Phone: 390-51-341652. Fax: 390-51-341632. E-mail: viroland{at}med.unibo.it.
| |
REFERENCES |
| 1.
|
Baldick, C. J., Jr., and T. Shenk.
1996.
Proteins associated with purified human cytomegalovirus particles.
J. Virol.
70:6097-6105[Abstract].
|
| 2.
|
Battista, M. C.,
G. Bergamini,
M. C. Boccuni,
F. Campanini,
A. Ripalti, and M. P. Landini.
1999.
Expression and characterization of a novel structural protein of human cytomegalovirus, pUL25.
J. Virol.
73:3800-3809[Abstract/Free Full Text].
|
| 3.
|
Bogner, E.,
M. Reschke,
B. Reis,
T. Mockenhaupt, and K. Radsak.
1993.
Identification of the gene product encoded by ORF UL56 of the human cytomegalovirus genome.
Virology
196:290-293[Medline].
|
| 4.
|
Dallas, P. B.,
P. A. Lyons,
J. B. Hudson,
A. A. Scalzo, and G. R. Shellam.
1994.
Identification and characterization of a murine cytomegalovirus gene with homology to the UL25 open reading frame of human cytomegalovirus.
Virology
200:643-650[Medline].
|
| 5.
|
Gibson, W.
1996.
Structure and assembly of the virion.
Intervirology
39:389-400[Medline].
|
| 6.
|
Hensel, G.,
H. Meyer,
S. Gartner,
G. Brand, and H. F. Kern.
1995.
Nuclear localization of the human cytomegalovirus tegument protein pp150 (ppUL32).
J. Gen. Virol.
76:1591-1601[Abstract/Free Full Text].
|
| 7.
|
Irmiere, A., and W. Gibson.
1983.
Isolation and characterization of a noninfectious virion-like particle released from cells infected with human strains of cytomegalovirus.
Virology
130:118-133[Medline].
|
| 8.
|
Landini, M. P.,
B. Severi,
G. Furlini, and L. Badiali-De Giorgi.
1987.
Human cytomegalovirus structural components: intracellular and intraviral localization of p28 and p65-69 by immunoelectron microscopy.
Virus Res.
8:15-23[Medline].
|
| 9.
|
Liu, B., and M. F. Stinski.
1992.
Human cytomegalovirus contains a tegument protein that enhances transcription from promoters with upstream ATF and AP-1 cis-acting elements.
J. Virol.
66:4434-4444[Abstract/Free Full Text].
|
| 10.
|
Meyer, H.,
A. T. Bankier,
M. P. Landini,
C. M. Brown,
B. G. Barrell,
B. Rüger, and M. Mach.
1988.
Identification and procaryotic expression of the gene coding for the highly immunogenic 28-kilodalton structural phosphoprotein (pp28) of human cytomegalovirus.
J. Virol.
62:2243-2250[Abstract/Free Full Text].
|
| 11.
|
Sanchez, V.,
P. C. Angeletti,
J. A. Engler, and W. J. Britt.
1998.
Localization of human cytomegalovirus structural proteins to the nuclear matrix of infected human fibroblasts.
J. Virol.
72:3321-3329[Abstract/Free Full Text].
|
| 12.
|
Schmolke, S.,
P. Drescher,
G. Jahn, and B. Plachter.
1995.
Nuclear targeting of the tegument protein pp65 (UL83) of human cytomegalovirus: an unusual bipartite nuclear localization signal functions with other portions of the protein to mediate its efficient nuclear transport.
J. Virol.
69:1071-1078[Abstract].
|
| 13.
|
Severi, B.,
M. P. Landini,
G. Cenacchi,
N. Zini, and N. M. Maraldi.
1992.
Human cytomegalovirus nuclear and cytoplasmic dense bodies.
Arch. Virol.
123:193-207[Medline].
|
| 14.
|
Spaete, R. R.,
R. C. Gehrz, and M. P. Landini.
1994.
Human cytomegalovirus structural proteins.
J. Gen. Virol.
75:3287-3308[Abstract/Free Full Text].
|
| 15.
|
Stefan, A.,
P. Secchiero,
T. Baechi,
W. Kempf, and G. Campadelli-Fiume.
1997.
The 85-kilodalton phosphoprotein (pp85) of human herpesvirus 7 is encoded by open reading frame U14 and localizes to a tegument substructure in virion particles.
J. Virol.
71:5758-5763[Abstract].
|
| 16.
|
Tooze, J.,
M. Hollinshead,
B. Reis,
K. Radsak, and H. Kern.
1992.
Progeny vaccinia and human cytomegalovirus particles utilize early endosomal cisternae for their envelopes.
Eur. J. Cell Biol.
60:163-178.
|
| 17.
|
Winkler, M., and T. Stamminger.
1996.
A specific subform of the human cytomegalovirus transactivator protein pUL69 is contained within the tegument of virus particles.
J. Virol.
70:8984-8987[Abstract].
|
Journal of Virology, July 1999, p. 6073-6075, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Chevillotte, M., Landwehr, S., Linta, L., Frascaroli, G., Luske, A., Buser, C., Mertens, T., von Einem, J.
(2009). Major Tegument Protein pp65 of Human Cytomegalovirus Is Required for the Incorporation of pUL69 and pUL97 into the Virus Particle and for Viral Growth in Macrophages. J. Virol.
83: 2480-2490
[Abstract]
[Full Text]
-
Hansen, S. G., Strelow, L. I., Franchi, D. C., Anders, D. G., Wong, S. W.
(2003). Complete Sequence and Genomic Analysis of Rhesus Cytomegalovirus. J. Virol.
77: 6620-6636
[Abstract]
[Full Text]
-
Dal Monte, P., Pignatelli, S., Zini, N., Maraldi, N. M., Perret, E., Prevost, M. C., Landini, M. P.
(2002). Analysis of intracellular and intraviral localization of the human cytomegalovirus UL53 protein. J. Gen. Virol.
83: 1005-1012
[Abstract]
[Full Text]
-
Liu, Y., Biegalke, B. J.
(2002). The Human Cytomegalovirus UL35 Gene Encodes Two Proteins with Different Functions. J. Virol.
76: 2460-2468
[Abstract]
[Full Text]
-
Bahr, U., Darai, G.
(2001). Analysis and Characterization of the Complete Genome of Tupaia (Tree Shrew) Herpesvirus. J. Virol.
75: 4854-4870
[Abstract]
[Full Text]
-
Lazzarotto, T., Varani, S., Gabrielli, L., Pignatelli, S., Landini, M. P.
(2001). The tegument protein ppUL25 of human cytomegalovirus (CMV) is a major target antigen for the anti-CMV antibody response. J. Gen. Virol.
82: 335-338
[Abstract]
[Full Text]
Top
Abstract
Text
