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J Virol, July 1998, p. 5661-5668, Vol. 72, No. 7
Department of Molecular Microbiology and
Immunology, Oregon Health Sciences University, Portland, Oregon
97201
Received 15 January 1998/Accepted 8 April 1998
Endothelial cells (EC) have been implicated as constituting an
important cell type in the pathogenesis of human cytomegalovirus (HCMV). Microvascular and macrovascular EC exhibit different
biochemical and functional properties depending on the organ of origin.
Phenotypic differences between microvascular and macrovascular EC may
alter the ability of these cells to support HCMV replication. In this study, we compared the replication of HCMV in primary macrovascular aortic EC (AEC) with that in brain microvascular EC (BMVEC). An examination of IE72, pp65, and gB viral antigen expression in BMVEC and
AEC by immunoflourescence revealed similar frequencies of infected
cells. Intracellular production of virus was 3 log units greater in
BMVEC than in AEC, while equal quantities of extracellular virus were
produced in both cell types. HCMV infection of BMVEC resulted in rapid
cellular lysis, while the virus was nonlytic and continuously released
from HCMV-infected AEC for the life span of the culture. An examination
of infected cells by electron microscopy revealed the formation of
abundant nucleocapsids in both AEC and BMVEC. However, significant
amounts of mature viral particles were only detected in the cytoplasm
of BMVEC. These observations indicate that levels of HCMV replication
in EC obtained from different organs are distinct and suggest that persistently infected AEC may serve as a reservoir of virus.
Human cytomegalovirus (HCMV)
establishes a lifelong persistence in the host after primary infection.
Although macrophages from the peripheral blood have recently been
identified as a site of HCMV latency in asymptomatically infected
individuals (21), another potential cell type latently or
persistently infected by HCMV is vascular endothelial cells (EC).
Studies of autopsy tissue from HCMV-seropositive transplant patients
have revealed that EC commonly harbor virus without obvious
cytopathology (14). However, additional studies have
indicated that EC constitute one of several cell types that exhibit
cytomegally in virus-infected tissues (6, 19, 24). An
understanding of the role of EC in HCMV disease has been complicated by
the detection of viral DNA in arterial specimens from seropositive
individuals without active infection (12). These
observations have led to the speculation that EC may be a virus
reservoir.
EC exhibit phenotypic differences that are dependent on the origin
(adult versus fetal), anatomical location, and vessel size (large
vessel versus capillary) (16, 22). Human umbilical vein EC
(HUVEC), which are commonly utilized for EC studies, are derived from
fetal large-vessel tissue. These cells are functionally and
biochemically distinct from EC derived from adult tissue such as
large-vessel aortic EC (AEC) (16, 22). Capillary EC not only
display unique differences from large-vessel EC but also demonstrate
organ specificity. For example, human brain microvascular EC (BMVEC),
which together with astrocytes compose the blood-brain barrier, possess
specific transporter systems that regulate the passage of specific
metabolites from the blood to the brain parenchyma (8, 13).
These unique properties differentiate BMVEC from EC in capillaries of
other tissues. The physiological and biochemical differences between EC
in different organs may affect the ability of HCMV to replicate in
these cells.
HCMV infection of EC in vitro has been controversial. Early studies
suggested that HCMV was unable to replicate in EC (4). However, others suggested that the virus could productively infect a
low percentage of cells in culture (20). In addition, HCMV infectivity of EC was enhanced by serial passage of virus through these cells. Interestingly, viral infection of HUVEC resulted in
anchorage-independent growth and a transformed phenotype
(23). An important consideration, however, is that viral
replication in HUVEC may not represent viral replication in adult EC.
In support of this hypothesis, other viruses have demonstrated
exquisite cellular specificity concerning their abilities to
productively infect EC obtained from different organs (13).
Lathey et al. addressed this issue when they demonstrated that HCMV
infected BMVEC more efficiently than HUVEC, suggesting that the
observed physiological differences between EC types may also affect
viral replication (10).
In the present study, we examined characteristics of HCMV replication
in AEC. We found that HCMV infection was not lytic and resulted in the
accumulation of significant amounts of extracellular but not
intracellular virus. In addition, the cell cycle was not inhibited by
HCMV and cells continuously released infectious virus. These results
contrast with those showing the rapid, lytic infection of BMVEC and HF
cells. The ability of HCMV to infect AEC and to continuously produce
extracellular virus and the absence of cytopathic effect are
prerequisites for establishing viral persistence. Therefore, HCMV
infection of AEC provides an ideal model to examine mechanisms of
persistence in the human host.
Culturing and infection of EC.
BMVEC were a generous gift
from Ashlee Moses (Oregon Health Sciences University, Portland, Oreg.),
and AEC were purchased from Clonetics Normal Human Cell Systems (San
Diego, Calif.). BMVEC were cultured in Endo-SFM medium (GIBCO
Laboratories, Grand Island, N.Y.) containing 10% human AB serum (Sigma
Chemical Co., St. Louis, Mo.), 1.0% penicillin-streptomycin solution
(GIBCO), 1.0% glutamine (Sigma), heparin (40 µg/ml; Sigma), and EC
growth factor (50 µg/ml; Sigma). AEC were cultured in the medium
recommended by the manufacturer. Since BMVEC and AEC cultures
represented single donors, each experiment was repeated in triplicate
with different donors. Low-passage-number (less than 5) BMVEC or AEC were plated in 35-mm2 Primaria culture dishes (Becton
Dickinson, Lincoln Park, N.J.) or two-well coverslip bottom Lab-Tek
chamber slides (Nunc, Inc., Naperville, Ill.) and allowed to grow at
37°C with 7% CO2 to 70% confluency prior to infection
with the HCMV laboratory strain Towne, a recent patient isolate Po
(3), or the mutant laboratory strain AD169-pp65
(17). Heparin-free medium was used at least 1 h prior
to infection and throughout the infection time course. Supernatants
from HCMV-infected HF cells were used as the source of the EC inoculum.
For mock infections, HF supernatant virus was UV inactivated for 8 h. The virus titer was measured on HF cells by a plaque assay as
previously described (3).
Immunofluorescence of HCMV IE and late antigens.
HCMV-infected EC grown on chamber slides were fixed for 20 min at room
temperature in buffered picric acid-paraformaldehyde (2%
paraformaldehyde and 15% buffered picric acid) and permeablized with
0.3% Triton X-100 in phosphate-buffered saline (PBS). Monolayers were
blocked with 20% normal goat serum in PBS and incubated for 1 h
at 37°C with one of the following antibodies raised against HCMV gene
products: a rabbit polyclonal immediate-early (IE)-specific antibody
(9), a monoclonal late antibody to pp65, or a monoclonal antibody against gB (1). Cell surface gB antigen was
detected prior to permeabilization of cells. The polyclonal von
Willebrand's factor (vWF) antibody was purchased from DAKO Corp.
(Carpinteria, Calif.). The binding of primary antibody was detected
with fluorescein isothiocyanate-, tetramethyl rhodamine isocyanate-, or
cyanine-5 (Biological Detection Systems, Inc., Pittsburgh,
Pa.)-conjugated secondary antibodies raised in the appropriate species
and visualized on an upright Leitz fluorescence microscope or a Leica
confocal laser scanning microscope equipped with a Leitz Fluorovert-FU microscope and an argon-krypton laser. The Slowfade antifade kit (Molecular Probes, Inc., Eugene, Oreg.) was utilized to ensure minimal
fluorescence fading.
FACS.
Subconfluent primary AEC cultures at passage 7 were
serum starved for 72 h. Starved cultures were mock infected or
infected with either AD169 or Towne HCMV at a multiplicity of infection (MOI) of 5, followed by an additional 12-h incubation period in serum-free media. During some experiments, phosphonoacetic acid (100 µg/ml) or 0.5 mM foscarnet was present in the media following infection. Cells were harvested both prior to adding serum to the
media, which was done at 12 h postinfection (hpi) and at intervals following serum addition. Trypsinized cells were rinsed once in PBS and
frozen at Tissue preparation for structural EM analysis.
To examine
virus-infected EC by electron microscopy (EM), uninfected and infected
cells were harvested at 3, 7, and 10 days after infection and fixed in
2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at 4°C
for 16 to 20 h. Fixed cells were collected by scraping and washed
in 0.15 M sodium cacodylate (pH 7.4). Specimens were postfixed in 1%
osmium tetroxide in the same buffer for 1 h at 4°C, dehydrated
in ethanol followed by acetone, and embedded in LX-112. Sections of
uninfected and infected cells on grids were washed and contrasted with
uranyl acetate-oxalate for 5 min, embedded in 2% methyl cellulose
containing 0.2% uranyl acetate, and examined with a calibrated Philips
420 electron microscope at 80 kV.
HCMV is lytic in BMVEC but not AEC.
Since AEC are naturally
infected in vivo and represent a potential reservoir of persistent
virus, we examined the ability of HCMV to infect primary cultures of
these large-vessel EC in vitro compared to the ability of the virus to
infect BMVEC. The EC cultures were >95% pure as determined by the
presence of vWF (Fig. 1A). AEC and BMVEC
subconfluent monolayers were infected with HCMV (Towne) at an MOI of 3 and examined at 3, 8, and 14 days postinfection (dpi) by phase
microscopy. HCMV infection of BMVEC resulted in the development of
cytopathic effect by 5 dpi and in lysis of 70% of the cells in culture
at 14 dpi (Fig. 1B and data not shown). Surprisingly, a cytopathic
effect was not observed in AEC infected with HCMV up to 30 dpi, the in
vitro life expectancy of these cells (Fig. 1B and
2B and data not shown).
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Human Cytomegalovirus Persistently Infects Aortic
Endothelial Cells
and
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
70°C in citrate-dimethyl sulfoxide (DMSO) buffer (40 mM
citrate trisodium, 5% DMSO, 250 mM saccharose), pH 7.6 (18). Nuclei were isolated in nucleus isolation buffer (10 mM Tris-HCl [pH 7.5], 1 mM MgCl2, 0.2 mM
phenylmethylsulfonyl fluoride, 0.25 M sucrose, 0.5% Triton X-100) for
30 min on ice with occasional vigorous vortexing, followed by a 5-min
spin at 14,000 rpm in an Eppendorf microcentrifuge at 4°C. This step
was repeated two to four times until greater than 80% of the nuclei
were clean. The nuclei were resuspended in 400 µl of citrate stock
solution (3.4 mM citrate trisodium, 0.1% IGEPAL CA-630 (Sigma), 1.5 mM spermine tetrahydrochloride, 0.5 mM Tris-HCl) (18). One-half of the nuclei were aliquoted into separate samples and stained with a
1:40 dilution of the polyclonal IE rabbit antibody described above,
followed by staining with a goat anti-rabbit antibody conjugated with
Cy2 (Amersham Life Sciences). All samples were resuspended in 200 µl
of the citrate stock solution; then 200 µl of a citrate stock
solution containing 250 µg of RNase A per ml was added, and the
samples were incubated for 10 min at room temperature. Finally, 100 µl of a citrate stock solution containing 2.1 mg of propidium iodide
per ml was added, and the samples were incubated for 30 to 60 min at
4°C prior to fluorescence-assisted cell sorting (FACS) analysis.
Becton Dickinson Cell Quest FACStation software (version 3.0.1)
operating a Becton Dickinson FACSCalibur FACS scan instrument was used
for analyzing the stained nuclei.
RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (126K):
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FIG. 1.
HCMV cytopathic effect in BMVEC and AEC. To ensure the
purity of EC, AEC and BMVEC were stained for the presence of vWF. As
shown in panel A, >95% of the cells displayed the presence of vWF
(magnification, ×125). Panel B demonstrates phase microscopy of
HCMV-infected AEC and BMVEC and HF cells at the indicated intervals
postinfection. As shown in this panel, cytopathic effect is observed by
8 dpi in BMVEC but not in AEC throughout the time course of infection
(magnification, ×50).
View larger version (23K):
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FIG. 2.
Expression of HCMV antigens in infected AEC and BMVEC.
(A) HCMV-infected AEC and BMVEC were examined by immunoflourescence to
assess the frequency of infected cells as well as the distribution of
viral antigens within the cell. Quantitation of the number of infected
cells indicated that at least 80% of the AEC and 50% of the BMVEC
expressed an HCMV antigen (IE72 or pp65). (B) To determine the
frequency of HCMV infection, double-label immunofluorescence was
performed with antibodies directed against the IE72 antigen (rhodamine;
red) and the pp65 antigen (fluorescein; green). Magnification, ×63.
(C) To determine the subcellular location of viral antigens, double-
and triple-label immunofluorescence was performed with antibodies
directed against the IE72 antigen (rhodamine; red), pp65 (BMVEC at 1 dpi as indicated by the arrow; fluorescein; green), or intracellular gB
antigen (fluorescein; green), and cell surface gB antigen (BMVEC at 3 dpi as indicated by the arrow; cyanine-5; blue). Magnification, ×265.
HCMV productively infects BMVEC and AEC. A one-step growth curve was generated from HCMV-infected AEC and BMVEC cultures to assess the abilities of these cells to support viral growth. An analysis of EC lysates revealed the production of significant quantities of cell-associated virus in BMVEC but not AEC (Fig. 3). Interestingly, similar amounts of virus were detected in supernatants obtained from both the BMVEC and AEC cultures (Fig. 3). These observations suggest that infectious intracellular virus produced in AEC is rapidly exported from infected cells in contrast to HCMV produced in infected BMVEC.
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HCMV infection of AEC does not inhibit the cell cycle. The ability of HCMV to nonlytically infect AEC and to produce infectious virus suggests that these cells may be a persistent reservoir of virus. HCMV-infected AEC did not differ in their phenotypic properties from mock-infected cells for up to 30 dpi in culture or when passaged (Fig. 6A and data not shown). The latter result suggests that HCMV does not inhibit the ability of AEC to progress through the cell cycle. To address this issue, subconfluent AEC cultures were serum starved for 48 h, followed by HCMV infection at an MOI of 5. At this MOI greater than 95% of the cells were positive by FACS for the IE antigen (data not shown). The ratio of infected to uninfected cells was similar up to 5 dpi (Fig. 6A). These observations indicate that HCMV does not alter the frequency of cell division in infected cells.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In this study we demonstrate that EC obtained from different adult tissues respond differentially to HCMV infection. Although both BMVEC and AEC are productively infected by virus, infection of BMVEC resulted in the rapid lysis of cells, while infected AEC cultures exhibited a sustained noncytopathic infection for over 30 dpi. Productive infection in the absence of cytopathology is a prerequisite for the establishment of persistence. Since HCMV infection of AEC in vitro is noncytopathic and since AEC are infected in vivo, this observation suggests that the large-vessel endothelium may be an HCMV reservoir in vivo.
The mechanism by which HCMV induces a persistent noncytopathic infection of AEC is unknown. HCMV-infected AEC were unable to accumulate infectious intracellular virus, although they produced normal amounts of extracellular virus. This phenomenon suggests that the cells survive infection through efficient export of mature virus and toxic products, which may cause cellular lysis, from the cell. This mechanism is clearly different from that which operates during HCMV infection of monocyte-derived macrophages, which results in the nonlytic accumulation of large amounts of intracellular virus and the lack of extracellular virus (3). In monocyte-derived macrophages, virus was found to accumulate in large cytoplasmic vacuoles that did not associate with the PM. These two observations emphasize the cell-specific differences that occur during HCMV infection.
In addition to the extremely rapid export of virus from AEC, the inability of HCMV to block the cell cycle may be a way in which the virus and cell coexist. The mechanism by which the cell or virus or both circumvent the normal blocks in the cell cycle due to infection is unknown. Jault et al. (7) observed an overexpression of cyclin E and a delay in cyclin A accumulation in HCMV-infected cells. Cyclin A is required for DNA replication and for the G2/M transition (5, 15). A proposed mechanism by which HCMV affected cyclin A accumulation was dependent upon viral and cellular DNA replication kinetics. Therefore, one might speculate that viral and cellular DNA replication kinetics in HCMV-infected AEC are such that the viral cell cycle inhibitors are not present at the appropriate times during the cell cycle. As the complexities of how HCMV affects the cell cycle are discovered, the mechanism by which AEC elude a block in the cell cycle may be revealed.
Although the specific cellular organs HCMV targets during acute disease have been known for almost 50 years, the site(s) for HCMV latency has been difficult to identify. We have recently demonstrated that latent HCMV can be reactivated in a myeloid lineage cell obtained from the peripheral blood of healthy seropositive individuals (21). However, this observation does not preclude the existence of other sites of HCMV latency or persistence. Another likely cellular candidate for HCMV persistence is the EC. AEC interact naturally with monocytes that are trafficking in the bloodstream and migrating into tissues. Therefore, a dynamic interaction between the cells in which virus is either activated or transmitted in tissues may occur during extravasation. These interactions may involve cell-cell contact through adhesion molecules, which subsequently triggers signal transduction events, activating virus in latently infected monocytes or the resting endothelium. The monocyte or AEC may function as a vehicle for cell-to-cell transmission of HCMV, which in turn could result in the reactivation of HCMV from macrophages or EC in the artery. One consequence of HCMV reactivation in the artery is the dissemination of virus throughout the body. HCMV would be easily spread throughout the circulatory system once shed into the lumen of the aorta because of the large quantities of blood that pass through this organ. The state of viremia may then result in the widespread infection of different organs. In addition to causing the dissemination of free virus throughout the body, circulating monocytes that become infected or that reactivate virus because of contact with AEC would serve as the ideal vector for HCMV dissemination to other tissues. In support of this hypothesis, an in vitro model has demonstrated that infected EC can transmit HCMV to cocultured monocytes and that these monocytes can retransmit virus to uninfected EC (23).
This study clearly indicates the importance of EC in the biology of HCMV. Elucidating the mechanisms of HCMV replication and virus assembly in AEC will be essential for understanding viral persistence and trafficking in the human host. The current studies indicate that HCMV replication in AEC differs from that in other naturally infected host cells. These observations emphasize the importance of examining viral replication in biologically relevant cell types.
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ACKNOWLEDGMENTS |
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We thank Rebecca Ruhl for technical assistance and Ashlee Moses for helpful discussion.
This work was supported by a Public Health Service grant from the National Institutes of Health (AI 21640) (J.A.N.), the Molecular Hematology Training program NIH NRSA Training Award (K.N.F.), and the Knut and Alice Wallenbergs Foundation (C.S.-N.). C.S.-N. is a scholar of the Wenner-Gren Foundation, Sweden.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, L220, 3181 S. W. Sam Jackson Park Rd., Portland, OR 97201. Phone: (503) 494-2434. Fax: (503) 494-6862. E-mail: nelsonj{at}ohsu.edu.
Present address: Karolinska Institute, Department for Biosciences
at Novum, Huddinge, Sweden.
Present address: Abteilung fur Medizinische Virologie,
Universität Tübingen, Tübingen, Germany.
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J Virol, July 1998, p. 5661-5668, Vol. 72, No. 7
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