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Journal of Virology, January 2000, p. 513-517, Vol. 74, No. 1
0022-538X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Human Cytomegalovirus Infects Caco-2 Intestinal
Epithelial Cells Basolaterally Regardless of the Differentiation
State
Audrey
Esclatine,1
Michel
Lemullois,2
Alain L.
Servin,1,*
Anne-Marie
Quero,1 and
Monique
Geniteau-Legendre1
Institut National de la Santé et de la
Recherche Médicale, Unité 510, Pathogènes et
Fonctions des Cellules Epithéliales Polarisées,
Faculté de Pharmacie, Université Paris XI, 92296 Châtenay-Malabry Cedex,1 and
Service Commun de Microscopie, Faculté d'Orsay,
Université Paris Sud, 91405 Orsay Cedex,2
France
Received 4 February 1999/Accepted 17 September 1999
 |
ABSTRACT |
Human cytomegalovirus (CMV) causes severe disease in
immunosuppressed patients and notably infects the gastrointestinal
tract. To understand the interaction of CMV with intestinal epithelial cells, which are highly susceptible to CMV infection in vivo, we used
the intestinal epithelial cell line Caco-2 and demonstrated that CMV
enters predominantly through the basolateral surface of polarized
Caco-2 cells. As shown by expression of all three classes of CMV
proteins and by visualization of nucleocapsids by transmission electron
microscopy, both poorly and fully differentiated Caco-2 cells were
permissive to CMV replication. However, infection failed to produce
infectious particles in Caco-2 cells, irrespective of the state of differentiation.
| |
TEXT |
Human cytomegalovirus (CMV) is a
ubiquitous virus that is a major cause of morbidity and mortality in
immunocompromised individuals. During severe CMV diseases in AIDS
patients, the gastrointestinal tract is frequently involved and is the
second main site of infection, after the retina (3, 8).
Although any location within the gut may be affected, gastrointestinal
CMV disease typically produces mucosal erosion or ulceration with
inflammation, tissue necrosis, and vascular injury (8). CMV
infects a broad spectrum of gastrointestinal cells, notably endothelial
cells, smooth muscle cells, macrophages, fibroblasts, and epithelial
cells of the mucosa (23, 25). Infection of the endothelial
cells can explain ischemic mucosal injury with concomitant necrosis.
However, epithelial cells are frequently infected, suggesting that they
may also be involved in the pathological process.
To further understand the role of intestinal epithelial cells in
gastrointestinal CMV disease, we investigated the ability of strain
AD169 of human CMV to initiate infection in the human intestinal
epithelial cell line Caco-2. Caco-2 cells differentiate spontaneously
in culture (6, 18), as they do in the normal intestine, and
exhibit many of the morphologic and functional properties of
enterocytes (28). The efficiency of CMV replication in other
cell systems appears to depend on the state of differentiation of the
cell (4, 7, 11, 20). Thus, to examine the relationship of
differentiation and infectibility, Caco-2 cultures grown on coverslips
were infected 3, 6, and 14 days postseeding. Before confluency, cells
are organized in islets. Peripheral cells of these islets are
undifferentiated and proliferate, whereas central cells become
polarized and then differentiate. After confluency, the whole monolayer
is polarized, but the process of differentiation requires many more
days to reach completion. At 14 days postseeding, cells were fully
differentiated, since we detected expression of sucrase-isomaltase, a
marker of differentiation, on the whole monolayer (data not shown)
(13). Cells were infected with the AD169 strain of CMV at a
multiplicity of infection (MOI) of 1, centrifuged for 45 min at
1,400 × g during the adsorption period to enhance
infectivity of CMV (10), fixed in acetone-water 48 h
postinfection (p.i.), and examined by immunofluorescence for the
intranuclear presence of the CMV immediate-early (IE) antigens with the
monoclonal antibody E13 (Biosoft).
Different patterns of infection were observed when Caco-2 cells were
infected with CMV at different ages. Cells grown for 3 days consisted
mainly of small heterogeneous-sized islets. In small islets, nearly all
cells showed IE proteins. In contrast, IE protein expression in larger
islets was restricted to their outer edges (Fig.
1A). A similar pattern was obtained with
monolayers grown for 6 days that consisted of large islets. After
infection, IE antigens were seen almost exclusively in cells at the
outer edges of the islets (Fig. 1B). Infection with CMV of 14-day-old Caco-2 monolayers showed that only a few cells expressed IE antigens (Fig. 1C).
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FIG. 1.
Cells were infected with CMV, fixed 48 h p.i., and
examined by indirect immunofluorescence with antibody clone E13,
directed against CMV IE proteins, and fluorescein
isothiocyanate-conjugated secondary antibody. (A) Infection of
3-day-old Caco-2 cell islets appeared to be restricted to the outer
edge. Six-day-old Caco-2 cells, which are subconfluent and consist of
large islets, were infected with CMV without any treatment (B) or
treated with EGTA (Sigma) (D). Fourteen-day-old Caco-2 cells grown on
glass coverslips infected with CMV that were untreated (C) or treated
with EGTA (E).
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Restriction of infection to the outer edges of islets raised the
possibility that CMV infects polarized Caco-2 cells preferentially through the basolateral surface. Indeed, in peripheral islet cells, the
virus has access to the entire cellular surface. In contrast, central
cells of the islet, which are already polarized and have tight
junctions, are accessible only by the apical domain.
Virus enters polarized Caco-2 cells preferentially through the
basolateral membrane domain.
We tested the polarity in the
infection process by applying virus either to the apical surface of
cells grown on porous filters (Transwell-clear; pore size, 0.4 µm;
Costar, Brumath, France) or to their basolateral surface.
Fourteen-day-old cells were infected with CMV at an MOI of 1 and
examined for expression of IE antigens by indirect immunofluorescence.
To centrifuge filters during the adsorption period, virus inocula must
be added in the upper reservoir of the filter. Cells were therefore
seeded on the lower side of the filter as previously described by
Perdomo et al. (Fig. 2) (17). Of
the differentiated Caco-2 cells infected with CMV through the apical
membrane and fixed 48 h p.i., only a small number expressed IE
antigens (Fig. 3A). In contrast, infection of
Caco-2 cells through the basolateral membrane led to a large number of
cells expressing CMV IE antigens (Fig. 3B). Figure 3C presents the
percentage of cells expressing IE antigens 24 h p.i. and shows
that the preference CMV displayed for the basolateral membrane was
17-fold.
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FIG. 2.
(A) Caco-2 cells were seeded onto inverted inserts to
allow centrifugation during basolateral infection. Cells were allowed
to attach overnight, after which the filters were placed upright in
12-well culture plates, thus orienting the basolateral side of the
monolayer upward. The apical (a) surfaces of monolayers were suspended
in the lower chamber. The CMV inoculum was placed in the upper chamber
for infection through the basolateral (bl) surface. (B) Apical
infection was performed on cells grown normally on filters. Adsorption
was carried out by centrifugation at 350 × g for 45 min. Cellular integrity was verified after centrifugation by measuring
lactate dehydrogenase activity and transepithelial resistance.
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FIG. 3.
Polarized entry of CMV into Caco-2 cells. Expression of
CMV IE antigens in polarized Caco-2 cells grown on porous filters
inoculated through either the apical (A) or basolateral (B) surface
with the AD169 strain at an MOI of 1 is shown. Only cultures with
transepithelial electrical resistance greater than 800  · cm2 were used. Cells were fixed 48 h p.i. and examined
by indirect immunofluorescence for expression of IE proteins. (C)
Infection of Caco-2 cells occurred essentially through the basolateral
membrane. (D) Expression of pp65 in 14-day-old Caco-2 cells cultured on
permeable filters infected with CMV at an MOI of 1 and fixed 6 h
p.i. We used monoclonal antibody 95/30, which reacts with the E
phosphoprotein pp65 and was a gift from Susan Michelson (Institut
Pasteur, Paris, France). This antibody can detect entry of virions and
dense bodies. A, apical infection; BL, basolateral infection.
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|
It is well known that tight junctions of polarized cells can be
disrupted by treating monolayers with EGTA, a Ca
2+
chelator, thus exposing the lateral pole of the cells (
19).
Such treatment has been used with Caco-2 cells to demonstrate
the
lateral entry of
Shigella flexneri, an enterovirulent
bacterium
(
16). We therefore investigated the effects of
disrupting tight
junctions by treatment with 100 µM EGTA on CMV entry
into Caco-2
cells grown on glass coverslips. Treatment with EGTA for
1 h prior
to infection and throughout the adsorption period caused
a significant
increase in the number of infected cells among 6-day-old
Caco-2
cells compared to those in untreated cultures. It abolished the
restricted expression of IE antigens, resulting in homogeneous
distribution (Fig.
1B and D). Identical treatment of 14-day-old
differentiated cells dramatically increased the number of cells
expressing IE antigens (Fig.
1C and
E).
To verify that the differential expression of IE antigens between
apical and basolateral infection reflects differences in
viral entry,
we used an antibody directed against pp65. Indeed,
pp65, a constituent
of virions and dense bodies, accumulates in
the nuclei of infected
cells immediately after virus-cell contact,
before the onset of viral
genome expression (
5). Caco-2 cells
cultured on permeable
filters were infected apically or basolaterally
with CMV at an MOI of
1. Cells were fixed 6 h p.i. and stained
with anti-pp65. Figure
3D
shows that basolateral infection resulted
in 100% pp65-positive cells
versus only 10.45% for apical
infection.
Taken together, these results strongly indicate that initiation of CMV
infection in epithelial polarized Caco-2 cells is preferentially
restricted to the basolateral membrane. This observation supports
a
clinical characteristic of intestinal CMV disease. A number
of
observations have clearly demonstrated that CMV is primarily
disseminated hematogenously (
24). Moreover, most symptomatic
CMV disease manifestations are due to reactivation of latent virus,
which is associated with viremia. In peripheral blood, infectious
virus
is predominantly cell associated; endothelial cells (
9)
and
the monocyte/macrophage system (
26), which are infected
by
CMV, may contribute to hematogenous dissemination of infection.
These
infected blood cells have access to the basolateral surface
of
epithelial cells and thus could transmit CMV to intestinal
epithelial
cells.
Comparison of the sequential expression of CMV antigens in 6- and
14-day-old Caco-2 cells and in fibroblasts.
Depending on the cell
type, CMV undergoes permissive or abortive infection. In permissive
infection, CMV proteins are sequentially expressed in three phases: IE,
early (E), and late (L). In abortive infection, viral expression is
limited to those proteins appearing before viral DNA synthesis. Caco-2
cells were used at various stages of differentiation. Six and
14-day-old Caco-2 cultures grown on glass coverslips and on permeable
filters, respectively, were infected with CMV at an MOI of 1. Expression of IE, E, and L antigens was studied by immunofluorescence
with monoclonal antibody clone E13 (Biosoft), CCH2 (Dako), and SL-20
(Biosoft) (1), directed against IE, E, and L antigens,
respectively. The results were compared with those for CMV-infected
MRC-5 fibroblasts.
Data obtained by immunofluorescence microscopy and flow cytometry
(
21) are summarized in Table
1.
Although the percentages
of Caco-2 cells expressing IE, E, and L
proteins were lower than
those of MRC-5 cells, the kinetics of
expression of CMV antigens
were similar to those observed in MRC-5
cells. From these experiments,
it was evident that both 6- and
14-day-old Caco-2 cells were permissive
for all phases of CMV gene
expression. The presence of the L protein,
which is expressed only
after viral DNA synthesis (
1), showed
that viral DNA
synthesis occurred in Caco-2 cells, irrespective
of the state of
differentiation.
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TABLE 1.
Comparison of the expression of CMV IE, E, and L proteins
in MRC-5 cells and 6- and 14-day-old Caco-2 cells
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CMV-infected Caco-2 cells produce nucleocapsids but no infectious
particles.
To gain further insight into the assembly and
maturation processes of CMV in Caco-2 cells, differentiated infected
cells grown on permeable filters were fixed 14 days after basolateral
infection (MOI of 1) and prepared for transmission electron microscopy, as previously described (14). Many nucleocapsids with
electron-translucent and electron-dense cores were observed in the
nuclei (Fig. 4A). Nucleocapsids were seen
acquiring an envelope during budding through the inner leaflet of the
nuclear membrane (Fig. 4B). However, no cytoplasmic particles or dense
bodies, previously described as composed of pp65 aggregates surrounded
by membranes (22), were observed in infected Caco-2 cells.
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FIG. 4.
Electron micrographs of basolaterally infected,
filter-grown Caco-2 cells 14 days p.i. Filters were fixed with
glutaraldehyde, postfixed with osmium, dehydrated in ethanol, and
embedded in epoxy resin. Thin sections of Caco-2 monolayers are shown
at different magnifications. (A) A nucleus containing numerous
nucleocapsids with a ring-shaped core (thick arrow) and a dense core
(thin arrow). A toroidal core was also visible (arrowhead). Important
morphological changes in nuclear structures that are characteristic of
late-stage CMV infection were observed. These changes consisted notably
of nuclear enlargement, condensation and margination of
heterochromatin, and irregularities of the nuclear outline, which was
frequently tortuous. (B) A nucleocapsid (thin arrow) acquiring an
envelope by budding through the inner leaflet of the nuclear membrane
was seen. N, nucleus; C, cytoplasm. Bars, 200 nm.
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Since many nucleocapsids with dense cores were observed, we asked
whether Caco-2 cells produce infectious particles. To analyze
whether
extracellular and cell-associated infectious virus was
produced, we
infected 6- and 14-day-old Caco-2 cells grown on
permeable filters.
Six-day-old Caco-2 cells were infected at an
MOI of 0.2 and 2, and
14-day-old cells were infected at an MOI
of 1. Titers of virus in
apical and basolateral media and cell
lysates collected at 2 to 24 days
p.i. were determined on MRC-5
cells. Neither extracellular nor
intracellular infectious virus
was found in Caco-2 cells, even 24 days
p.i., irrespective of
the state of differentiation at the time of
infection.
Although IE, E, and L antigens were expressed in Caco-2 cells, this did
not correlate with virus production in either differentiated
or
undifferentiated cells. This is in striking contrast to the
results
described by Jarvis et al. (
12), published after our
paper
was originally submitted. They showed that basolateral infection
of
Caco-2 cells is productive, with progeny virus localized
intracellularly
and released from the apical membrane domain. This
difference
may be related to the CMV strains used. Indeed, we used the
AD169
strain, whereas they used another laboratory strain, the Towne
strain. It has been reported that infection of differentiated
THP-1
monocytes with the Towne strain, as opposed to the AD169
strain,
results in productive infection (
27). Another explanation
may be the very high MOI (a total of 25) used by Jarvis et al.
to
infect Caco-2 cells, which still allowed only a low level of
production. After infection with the Towne strain, progeny virus
was
released from the apical membrane domain, which was consistent
with an
apical localization of an essential glycoprotein, gB.
Upon infection by
the AD169 strain, expression of gB was observed
only within
permeabilized cells in our laboratory studies, suggesting
intracellular
sequestration of this protein (data not shown).
This correlates with
the observed lack of virus production. To
gain further insight into the
assembly and maturation processes
of CMV in Caco-2 cells, electron
microscopic analyses were performed.
Many nucleocapsids were observed
in the nucleus and in the perinuclear
space, while neither virus
particles nor dense bodies were seen
in the cytoplasm. The absence of
dense bodies may be related to
the low level of pp65 expression we
observed after 24 days of
infection (data not shown). This again
correlates with the nonproductive
state.
Other differences between the AD169 and Towne strains can be noted.
Infection of Caco-2 cells with the AD169 strain at an
MOI of 1 leads to
percentages of cells expressing CMV IE, E, and
L proteins similar to
the percentages obtained with the Towne
strain at an MOI of 25 (
12). After 4 days of infection, we observed
about 40%
IE-positive cells and 5% E- and L-positive cells. Similar
percentages
of expression were observed by Jarvis et al. at 8
days p.i. for IE
proteins and at 12 days p.i. for E and L proteins.
Surprisingly, in
their study, Jarvis et al. detected L protein
after the appearance of
intracellular and extracellular infectious
virus.
Although CMV does not seem to replicate in Caco-2 cells, it is well
known that CMV can modify and damage cells in the absence
of a complete
replicative cycle (
2). It would thus be of interest
to study
whether Caco-2 cells are altered functionally and/or
morphologically by
CMV
infection.
Finally, it was reported that, in vitro, the efficiency of CMV
replication seems to depend on the state of cellular differentiation
(
4,
20). However, we showed here that infection with CMV
of
Caco-2 cells is independent of their state of differentiation.
Jarvis
et al. (
12) failed to infect fully differentiated Caco-2
cells, but this is perhaps related to the laboratory strain used.
Laboratory strains of CMV have deletions in their viral genomes
compared to those in clinical isolates (
15). Subsequently,
it
will be important to compare infectivity of the laboratory strain
AD169 with that of fresh clinical
isolates.
| |
ACKNOWLEDGMENTS |
We are very grateful to Susan Michelson for helpful discussions and
assistance in the preparation of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: INSERM U-510,
Faculté de Pharmacie, 5 rue J. B. Clément, 92296 Châtenay-Malabry Cedex, France. Phone and fax: 33 1 46 83 56 61. E-mail: alain.servin{at}cep.u-psud.fr.
| |
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Journal of Virology, January 2000, p. 513-517, Vol. 74, No. 1
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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