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Journal of Virology, September 1998, p. 7598-7602, Vol. 72, No. 9
Department of Laboratory
Medicine1 and
Department of Obstetrics
and Gynecology,
Received 13 April 1998/Accepted 20 May 1998
Human trophoblast cells were permissively infected by human
cytomegalovirus. The kinetics of viral immediate-early, early, and late
gene expression was clearly delayed compared to that in fibroblasts.
Productive infection was unequivocally proven by the detection of
virion particles, infectious virus in trophoblast culture supernatant,
and cell-to-cell spread of cytomegalovirus from infected trophoblasts
to uninfected fibroblasts. These observations indicate that infected
trophoblasts may be involved in maternofetal transmission of human
cytomegalovirus.
Maternofetal transmission of human
cytomegalovirus (HCMV) is the most common cause of congenital viral
infection. The individual course of infection may vary between
asymptomatic virus shedding, long-term sensorineural deficits, abortion
or stillbirth, or congenital CMV syndrome, including thrombocytopenia,
hepatosplenomegaly, and mental retardation (1). The factors
determining the pathogenesis of this infection are widely unknown.
Interestingly, in the murine system, the conditions of primary
infection in the organism seem to influence the subsequent course of
CMV infection (5). Thus, the mode of transplacental virus
transmission during initial maternal viremia might be an important step
in the pathogenesis of the congenital HCMV syndrome by determining the
viral load in the fetal organism. In this context, a cell type of major
interest is the trophoblast, which forms the interface between maternal blood and fetal tissue. In a guinea pig model, productive infection of
the syncytiotrophoblast was found (3), which suggested that human trophoblasts might also be a target of HCMV infection. Actually, HCMV infection of the trophoblast has been described in placental tissue from congenitally infected fetuses (4, 8). However, productive infection of the trophoblast has not yet been demonstrated. Viral gene expression seemed to be restricted to immediate-early or
early gene products (8, 9). In a recent study, late viral proteins were found in trophoblasts only after human immunodeficiency virus (HIV) coinfection (9), a situation that is absent in the vast majority of natural maternofetal HCMV transmissions. In the
present study, we demonstrate by several lines of evidence that
trophoblast cells can be permissively and productively infected by HCMV
without any coinfecting agent.
Infection of trophoblast cultures with HCMV.
Trophoblast cells
were isolated from human term placentae by trypsin digestion, percoll
gradient centrifugation, and immunoselection for HLA class I-negative
cells as described elsewhere (2, 7). Cells were seeded and
kept without further passaging in keratinocyte growth medium (Gibco,
Eggenstein, Germany) supplemented with 20% fetal calf serum,
glutamine, gentamicin, recombinant epidermal growth factor, and bovine
pituitary extract. Cell cultures consisted of >95% trophoblasts as
judged by the immunodetection of the trophoblast marker proteins human
chorionic gonadotropin Delayed kinetics of viral antigen expression in trophoblast
cells.
Previous workers had suggested that trophoblast cultures
were nonpermissive for the full replicative cycle of HCMV (6, 9). Recently, HCMV gene expression was reported to be restricted to immediate-early and early genes. Only after coinfection with HIV did
these authors find unrestricted HCMV replication in trophoblast cultures (9). As abortion of HCMV infection after efficient early gene expression seems unlikely, we assumed that delayed kinetics
of viral gene expression might account for the difficulties in
detecting late viral gene products. Therefore, we analyzed immediate-early, early, and late gene expression in trophoblast cultures at various intervals after infection with cell-free
preparations of strain AD169 at an MOI of 1. In particular, we detected
the IE1 and IE2 proteins (UL122/123; MAb E13; Biosoft, Paris, France), the early protein p52 (pUL44; MAb BS510; Biotest, Dreieich, Germany), and the late major capsid protein (pUL86; MAb 28-4, kindly provided by
W. Britt) in acetone-fixed cells by an indirect immunoperoxidase technique (Fig. 2). Proteins of all
phases of HCMV replication were detectable in up to 100% of cells.
Characteristic cytopathogenic effects occurred, and the cell cultures
were completely lysed 14 days after infection. However, the kinetics of
viral gene expression was clearly delayed compared with that for
standard fibroblast cultures. We detected immediate-early antigens at 2 days postinfection (p.i.), early antigen at 4 days p.i., and late
antigen at 6 days p.i., except for very few cells that had slightly
faster kinetics (Fig. 2). Viral antigens were never detected in
mock-infected cultures. From these experiments, it was evident that
trophoblast cells were permissive to all phases of HCMV gene expression
and that infection was cytopathogenic and lytic.
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Human Trophoblast Cells Are Permissive to the
Complete Replicative Cycle of Human Cytomegalovirus
ABSTRACT
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(beta-hCG; antibody from Dako, Hamburg,
Germany) and cytokeratin 8/18/19 (antibody from Monosan, Am Uden, The
Netherlands). Below 5% were stromal cells expressing the mesenchymal
cell marker vimentin (antibody from Dako). We first analyzed the
susceptibility of these cells for various HCMV strains, including three
clinical HCMV isolates and HCMV laboratory strains AD169 and Towne.
Trophoblast cultures in 96-well plates were infected with cell-free
virus preparations at multiplicities of infection (MOIs) between 0.1 and 1. After 48 h of infection, HCMV immediate-early antigen was
detected in the nuclei of infected cells by immunostaining using
monoclonal antibody (MAb) E13 (anti-pUL122/123). Infected cells were
identified as trophoblasts by the detection of trophoblast marker
beta-hCG (Fig. 1). With either HCMV
strain, between 5 and 50% of cells were infected. This indicated that
trophoblast cultures were readily susceptible to HCMV infection,
irrespective of the virus strain used.
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FIG. 1.
Multinucleated trophoblast cell 5 days after infection
with HCMV strain AD169 at an MOI of 1. (A) Phase-contrast micrograph.
Magnification, ×320. (B) Double immunofluorescence staining to detect
HCMV immediate-early antigens (FITC) and trophoblast marker beta-hCG
(TRITC). Magnification, ×320.
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FIG. 2.
Kinetics of HCMV gene expression in infected
trophoblasts. Detection of immediate-early antigen (MAb E13, UL122/123)
(A), early antigen p52 (MAb BS510, UL44) (B), and late antigen MCP (MAb
28-4, UL86) (C) in multinucleated trophoblast cells 6 days after
infection, by the indirect immunoperoxidase technique. (D) Time course
of antigen expression in infected trophoblast cells. d, days.
Infected trophoblast cells produce infectious CMV. Finally we asked whether late-stage-infected trophoblasts produce infectious HCMV particles. First, electron microscopic analyses were performed to visualize virion particles in late-stage-infected cells. Two days after isolation, trophoblast cultures were infected with HCMV strain AD169 at an MOI of 1 and cultured for an additional 6 days. Cultures were then prepared for transmission electron microscopy and examined at magnifications of 12,000 to 20,000. In multinucleated syncytiotrophoblast cells, we detected alterations of the nuclear ultrastructure that are characteristic of late-stage CMV infection. In so-called replication compartments, numerous herpesvirus capsids with or without DNA were visible (Fig. 3A). Coated particles at a lower frequency were detectable in the cytoplasm of these cells (data not shown). These ultrastructural data demonstrated the formation of HCMV particles in late-stage-infected trophoblasts.
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80°C for determination of the
infectious titer. Titers were determined by limiting dilution analysis
in fibroblast cultures. Single-step growth curves in infected
fibroblast cultures displayed viral replication beginning at day 2 p.i., with a peak at day 5 p.i. In contrast, production of
infectious HCMV in infected trophoblast cultures was delayed, beginning
at day 5 p.i. and peaking at day 11 p.i. The titer was 3 logs
lower than that in identically infected fibroblast cultures; however,
HCMV was clearly detectable in three independent experiments. This
result strongly indicated that infectious virus was released from
late-stage-infected trophoblast cultures.
To formally prove that late-stage-infected trophoblast cells can
transmit infectious HCMV to adjacent cells, we performed a modified
infectious center assay (Fig. 3C). Freshly isolated trophoblast cells
were seeded sparsely on 6-well culture plates and infected with HCMV
strain AD169 at an MOI of 1. Five days after infection, noninfected
fibroblasts were seeded into the same culture, resulting in coculture
of a few infected trophoblasts with abundant noninfected fibroblasts.
After an additional day of coculturing, cells were fixed and stained
for cell marker proteins and viral antigens to detect whether
late-stage-infected trophoblasts were capable of transmitting HCMV to
adjacent fibroblasts.
The simultaneous detection of cellular and viral proteins was done by a
quadruple staining procedure, combining immunofluorescence and
immunocytochemistry as follows: (i) detection of HCMV major capsid
protein (MAb 28-4) by the PAP technique (Dako) with diaminobenzidine as
a chromogen, resulting in brown nuclear staining; (ii) detection of
HCMV immediate-early proteins (MAb E13) by the APAAP technique (Dako)
with fast blue as a chromogen, resulting in blue nuclear staining;
(iii) detection of mesenchymal marker vimentin (MAb from Dako) by
indirect fluorescein isothiocyanate (FITC) immunofluorescence, resulting in green cytoplasmic fluorescence; and (iv) detection of
trophoblast marker beta-hCG (polyclonal antibody from Dako) by
indirect tetramethyl rhodamine isothiocyanate (TRITC)
immunofluorescence, resulting in red cytoplasmic
fluorescence. The TRITC-conjugated anti-rabbit immunoglobulin
antibodies of step 4 additionally bound to the secondary rabbit
antibodies of step 2, thus yielding red nuclear fluorescence of
HCMV-infected cells, which could easily be distinguished from the
cytoplasmic trophoblast staining of step 4.
In three independent experiments we could consistently detect
infectious foci in the infected trophoblast-noninfected fibroblast cocultures. In the center of these foci we regularly found a
trophoblastic cell, as identified by beta-hCG staining. This
trophoblast expressed late viral structural antigen, while the adjacent
fibroblasts expressed only viral immediate-early antigen. This assay
unequivocally proved that late-stage-infected trophoblast cells can
transmit infectious HCMV to adjacent mesenchymal cells.
In summary, our results demonstrate the permissive productive infection
of trophoblast cells by HCMV. It is noteworthy that the kinetics of
gene expression was delayed in all phases of viral replication at least
twofold compared with that in standard fibroblast cultures. Our
infected trophoblast-noninfected fibroblast coculture model resembles
an in vivo situation that might occur during transplacental transmission of HCMV, thus supporting the hypothesis that infected trophoblasts may play a major role in maternofetal transmission of
HCMV.
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ACKNOWLEDGMENTS |
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This work was supported by grant P10900 (to G.D.) from the Austrian Research Foundation, by the Fortüne Projekt Tübingen (F.1432060.2), and by the IKFZ Tübingen (01KS9602).
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FOOTNOTES |
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* Corresponding author. Mailing address: Abt. Med. Virologie, Universität Tübingen, Calwerstrasse 7/6, D-72076 Tübingen, Germany. Phone: 49 7071 2987459. Fax: 49 7071 295790. E-mail: christian.sinzger{at}med.uni-tuebingen.de.
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Journal of Virology, September 1998, p. 7598-7602, Vol. 72, No. 9
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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