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10.1128/JVI.74.15.6808-6820.2000.
2000, 74(15):6808. DOI:J. Virol.
Pereira
Susan Fisher, Olga Genbacev, Ekaterina Maidji and Lenore
Pathogenesis
Utero: Implications for Transmission and
Placental Cytotrophoblasts In Vitro and In
Human Cytomegalovirus Infection of
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JOURNAL OF VIROLOGY,
0022-538X/00/$04.00⫹0
Aug. 2000, p. 6808–6820 Vol. 74, No. 15
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Human Cytomegalovirus Infection of Placental Cytotrophoblasts
In Vitro and In Utero: Implications for Transmission
and Pathogenesis
SUSAN FISHER,
1,2,3,4,5
* OLGA GENBACEV,
1
EKATERINA MAIDJI,
1
AND LENORE PEREIRA
1
*
Departments of Stomatology,
1
Obstetrics, Gynecology and Reproductive Sciences,
2
Anatomy,
3
and Pharmaceutical
Chemistry
4
and the Biomedical Sciences Graduate Program,
5
University of California San Francisco,
San Francisco, California 94143
Received 8 March 2000/Accepted 28 April 2000
Human cytomegalovirus (CMV) is the leading cause of prenatal viral infection. Affected infants may suffer
intrauterine growth retardation and serious neurologic impairment. Analysis of spontaneously aborted con-
ceptuses shows that CMV infects the placenta before the embryo or fetus. In the human hemochorial placenta,
maternal blood directly contacts syncytiotrophoblasts that cover chorionic villi and cytotrophoblasts that
invade uterine vessels, suggesting possible routes for CMV transmission. To test this hypothesis, we exposed
first-trimester chorionic villi and isolated cytotrophoblasts to CMV in vitro. In chorionic villi, syncytiotropho-
blasts did not become infected, although clusters of underlying cytotrophoblasts expressed viral proteins. In
chorionic villi that were infected with CMV in utero, syncytiotrophoblasts were often spared, whereas cytotro-
phoblasts and other cells of the villous core expressed viral proteins. Isolated cytotrophoblasts were also
permissive for CMV replication in vitro; significantly, infection subsequently impaired the cytotrophoblasts’
ability to differentiate and invade. These results suggest two possible routes of CMV transmission to the fetus:
(i) across syncytiotrophoblasts with subsequent infection of the underlying cytotrophoblasts and (ii) via
invasive cytotrophoblasts within the uterine wall. Furthermore, the observation that CMV infection impairs
critical aspects of cytotrophoblast function offers testable hypotheses for explaining the deleterious effects of
this virus on pregnancy outcome.
Human cytomegalovirus (CMV) infection, which usually has
a benign course in immunocompetent individuals, can have
catastrophic consequences during pregnancy (3). Primary
CMV infection during gestation poses a 30 to 40% risk of
intrauterine transmission and clinical disease (58, 59). Reacti-
vated infection is associated with at least a 10-fold-lower rate
of transmission. Congenital CMV infection is a relatively com-
mon occurrence, as approximately 1 to 4% of newborns in the
United States and Europe are infected with CMV (3), and
transmission could be higher in developing countries (13).
Many infected infants show no clinical manifestations of the
congenital CMV syndrome. Symptomatic infants often suc-
cumb in the neonatal period (12%), and most survivors have
permanent debilitating sequelae, including mental retardation,
vision loss, and sensorineural deafness. Since CMV establishes
latent infections in granulocyte-dendritic progenitors (25, 34,
56), the fetus may also become infected after reactivation of
maternal infection, a scenario that is usually associated with
less severe clinical disease in the offspring (18, 59). CMV
seroconversion rates and restriction endonuclease analyses of
virus strains indicate that heterosexual activity (5, 6, 17, 27) and
contact with young children (30, 47) are the major modes of
virus dissemination in women of childbearing age.
Despite the morbidity and mortality associated with prenatal
CMV infection, little is known about how the virus infects the
conceptus. Approximately 15% of women with primary infec-
tions during early pregnancy abort spontaneously (24). In this
case the placenta, but not the fetus, shows evidence of infec-
tion, which suggests that placental involvement is important in
its own right and precedes virus transmission to the fetus (1, 28,
44). Later in pregnancy CMV infection causes premature de-
livery and, in 25% of affected infants, intrauterine growth re-
tardation (31), outcomes that are often associated with placen-
tal pathology. Numerous reports indicate that placentas from
these births also contain viral proteins (44, 45), suggesting that
placental infection and virus transmission to the infant are
related causally.
An important role for the placenta in CMV transmission to
the fetus is also suggested by the unusual anatomy of the
maternal-fetal interface (Fig. 1), which is determined in large
part by placental development (reviewed in references 9 and
10). Placentation is a stepwise process that entails differentia-
tion of the organ’s specialized epithelial stem cells, termed
cytotrophoblasts. Two pathways give rise to the differentiated
trophoblast cells that are found in floating and anchoring cho-
rionic villi. In the pathway that gives rise to floating villi, cy-
totrophoblasts differentiate by fusing into multinucleate syncy-
tiotrophoblasts that cover the villous surface, where they are in
direct contact with maternal blood. This trophoblast popula-
tion is specially adapted for transporting a wide variety of
substances to and from the embryo or fetus. In the pathway
that gives rise to anchoring villi, cytotrophoblasts remain as
single cells that aggregate into columns and invade the endo-
metrium and the first third of the myometrium (interstitial
invasion). They also breach the portions of maternal arterioles
that span these regions (endovascular invasion). By midgesta-
tion, the latter population of cells completely replaces the
endothelial lining and much of the smooth muscle wall of these
vessels. The result is a hybrid vasculature composed of fetal
and maternal cells.
* Corresponding author. Mailing address: Department of Stomatol-
ogy, HSW-604, University of California San Francisco, 513 Parnassus
Ave., San Francisco, CA 94143-0512. Phone: (415) 476-5297 (S.F.),
(415) 476-8248 (L.P.). Fax: (415) 502-7338. E-mail: sfisher@cgl.ucsf
.edu or pereira@itsa.ucsf.edu.
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FIG. 1. (A) Diagram of a longitudinal section that includes a floating and an anchoring chorionic villus at the fetal-maternal interface near the end of the first
trimester of human pregnancy (10 weeks of gestational age) (modified from references 10 and 66). The anchoring villus (AV) functions as a bridge between the fetal
and maternal compartments, whereas the floating villus (FV), containing macrophages (MØ, Hofbauer cells) and fetal blood vessels, is bathed by maternal blood.
Cytotrophoblasts in AV (zone I) form cell columns that attach to the uterine wall (zones II and III). Cytotrophoblasts then invade the uterine interstitium (decidua
and first third of the myometrium; zone IV) and maternal vasculature (zone V), thereby anchoring the fetus to the mother and accessing the maternal circulation. Zone
designations mark areas in which cytotrophoblasts have distinct patterns of stage-specific antigen expression, including integrins and HLA-G. Decidual granular
leukocytes (DGLs) and macrophages (MØ) in maternal blood and fetal capillaries in villous cores are indicated in panels A and B. Areas proposed as sites of natural
CMV transmission to the placenta in utero are numbered 1, 2, and 3. (B) Diagram of a uterine (spiral) artery in which endovascular invasion is in progress (10 to 20
weeks of gestation). Endometrial and then myometrial segments of spiral arteries are modified progressively. In fully modified regions (a), the vessel diameter is large.
Cytotrophoblasts (CTBs) are present in the lumen and occupy the entire surface of the vessel wall. A discrete muscular layer (tunica media) is not evident. (b) Partially
modified vessel segments. Cytotrophoblasts and maternal endothelium occupy discrete regions of the vessel wall. In areas of intersection, cytotrophoblasts appear to
lie deep in the endothelium and in contact with the vessel wall. (c) Unmodified vessel segments in the myometrium. Vessel segments in the superficial third of the
myometrium will become modified when endovascular invasion reaches its fullest extent (about midgestation), while deeper segments of the same artery will retain their
normal structure.
VOL. 74, 2000 CMV INFECTION OF PLACENTAL CYTOTROPHOBLASTS 6809
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These unusual cell-cell interactions are the result of an
equally unusual molecular differentiation program. For exam-
ple, syncytiotrophoblasts that cover floating villi upregulate
expression of the neonatal immunoglobulin G (IgG) Fc recep-
tor (hFcRn), which binds and transports maternal IgG to the
fetus (38, 53, 62). This important process establishes passive
immunity to certain infectious agents. Invading cytotropho-
blasts that are components of anchoring villi switch on the
expression of adhesion molecules (e.g., integrin ␣11) and
proteinases (e.g., matrix metalloproteinase-9) that are needed
for invasion, as well as molecules that elicit maternal immune
tolerance (e.g., the nonclassical major histocompatibility com-
plex [MHC] class Ib molecule HLA-G [35, 43]) and the cyto-
kine interleukin-10 [51]). In a process termed pseudovasculo-
genesis, invading cells also transform their adhesion receptor
phenotype to resemble that of the endothelial cells that they
replace. For example, they express ␣v3 integrin, a marker of
angiogenic endothelium, and vascular endothelial cadherin
(10). Both cytotrophoblast invasion and pseudovasculogenesis
are essential for normal pregnancy, as serious complications
(e.g., preeclampsia) can occur when this process fails (41, 48,
64, 65).
A great deal of information about the human placenta,
largely inaccessible for study in utero, has been obtained by
studying culture models of the trophoblast populations that lie
at the maternal-fetal interface. The two most commonly used
models are villous explants and isolated cytotrophoblasts (15,
16, 20, 40). Explants (Fig. 2A) are essentially organ cultures of
anchoring villi in which cell columns attach to, and subse-
quently invade, an extracellular matrix substrate. When iso-
lated cells are plated on extracellular matrixes (Fig. 2B), they
rapidly differentiate along the invasive pathway, acquiring the
specialized properties of the cytotrophoblast subpopulation
that is found within the uterine wall. Both models have been
integral to recent progress made in understanding the factors
that govern assembly of the human maternal-fetal interface in
normal pregnancy and how this process goes awry in pregnancy
complications such as preeclampsia (10).
Here we used these culture models to study CMV infection
of human placental cells in vitro. During the course of these
experiments, we discovered that a significant number of pla-
centas had already been infected with CMV in utero, allowing
a rare glimpse into the natural process. This also gave us an
interesting opportunity to compare the populations of placen-
tal cells that expressed viral proteins in the two situations. We
also investigated the consequences of infection in vitro on the
ability of isolated cytotrophoblasts to differentiate along the
invasive pathway. We found that the placenta is not an effective
barrier to CMV transmission. Rather, cytotrophoblasts in sev-
eral locations become infected, suggesting specific routes by
which the virus reaches the fetus in utero. Furthermore, cy-
totrophoblasts are not a passive conduit: CMV infection re-
sulted in significant deficits in their ability to differentiate and
invade. Together, the results of these experiments suggest an
explanation for the association between CMV infection of the
fetus and intrauterine growth retardation, as well as strategies
for blocking the routes of transmission that we identified.
MATERIALS AND METHODS
Chorionic villus isolation and explant culture. Filters (12-mm diameter) with
0.4-m pores (Millipore Products Division, Bedford, Mass.) were coated with
100 l of Matrigel (Collaborative Research, Bedford, Mass.) as previously de-
scribed (20). Six- to eight-week human placentas were obtained from donors who
had normal pregnancies prior to termination. Approval for this project was
FIG. 2. Culture models for studying CMV infection of anchoring villus explants and differentiating cytotrophoblasts (CTBs). (A) Diagram of an anchoring villus
explant attached to a Matrigel substrate via cytotrophoblasts that migrate from the cell columns. (B) Diagram of purified cytotrophoblasts cultured on Matrigel. The
cytotrophoblast stem cells aggregate, invade the matrix, and express stage-specific molecules, including integrins and HLA-G. Cultured cytotrophoblasts mimic the
differentiation phenotype and morphology of cell columns formed in placentas in utero. For infection, CMV is added to the medium bathing the explants and
cytotrophoblasts.
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obtained from the Institutional Review Board at the University of California San
Francisco. Anchoring villi were dissected from placentas and transferred to the
coated filters. Cultures established from 13 placentas were used in this study.
Initially, 22 fragments containing tree-like anchoring villi were dissected from the
entire surface of each placenta. Ten were immediately fixed and processed for
immunolocalization studies as previously described (11). The remaining 12 were
cultured on Matrigel substrates in Dulbecco’s modified Eagle’s medium-F12
medium (DMEM-F12; 1:1, vol/vol; GIBCO, Rockville, Md.) supplemented with
10% fetal calf serum. After 12 h, six were infected with CMV as described below.
Explants were maintained for up to 96 h. This model system is diagrammed in
Fig. 2A.
Isolation and culture of purified cytotrophoblasts. Highly purified cytotropho-
blasts were isolated from 10- to 16-week placentas as previously described (40).
A small fraction of the cells (5 ⫻ 10
4
) were immobilized on slides by centrifu-
gation (Cytospin Cell Preparation System; Shandon Inc., Pittsburgh, Pa.) and
then fixed and stained with monoclonal antibody (MAb) CH160-5 to CMV
immediate-early proteins 1 and 2 (IE1/2) (14). The results showed whether
cytotrophoblasts were infected in utero. The remainder were resuspended in
DMEM containing 2% Nutridoma (Boehringer Mannheim Corp., Indianapolis,
Ind.). Transwell filters (6.5 mm in diameter, 5-m pore size; Costar, Corning,
N.Y.) were coated with 10 l of Matrigel, and then 2.5 ⫻ 10
5
to 5.0 ⫻ 10
5
cells
were plated on each. After 12 h, half the cultures were infected with virus as
described below. Cultures were maintained for up to 96 h. This model system is
diagrammed in Fig. 2B.
CMV stock viruses, infection, and titration. The construction of CMV(AD169)
mutants RV798 and RV670 with deletions in genes that downregulate expression
of classical MHC class I molecules has been published (33). Stock viruses were
prepared in human foreskin fibroblasts (HFF) grown in roller bottles, and the
infectivity titers were determined by immunofluorescence using a rapid infectiv-
ity assay (46). At 12 h after plating, villous explants and purified cytotrophoblasts
were infected with 10
6
PFU per filter. To count CMV progeny virions, cytotro-
phoblasts (2.5 ⫻ 10
5
to 5.0 ⫻ 10
5
) and HFF (0.5 ⫻ 10
5
to 1.0 ⫻ 10
5
) were plated
on Matrigel-coated filters (0.4-m pores) and infected with CMV(AD169) at 10
and 1 PFU/cell, respectively. At 24-h intervals, cells were harvested, sonicated to
release intracellular virus, and centrifuged at low speed to remove cell debris.
Released virions in the culture medium were counted separately.
Antibodies. A mouse MAb, CH160-5, to the CMV IE1/2 proteins was pro-
duced in the Pereira laboratory (14) and obtained as purified IgG from the
Goodwin Institute (Plantation, Fla.). Guinea pig antiserum to CMV gB (UL55)
was a generous gift from Chiron Corporation (Emeryville, Calif.). The following
antitrophoblast antibodies were produced in the Fisher laboratory unless other-
wise noted: a rat MAb, 7D3, to cytokeratin (11); a mouse MAb, 4H84, to a
synthetic peptide of the ␣1 domain of HLA-G (42); a mouse MAb, BIIG2, to
integrin ␣5; and anti-VLA-1 to integrin ␣1 (T-Cell Sciences, Cambridge, Mass.).
The specificities of the secondary antibodies, all of which were obtained from
Jackson ImmunoResearch Laboratories Inc. (West Grove, Pa.), were as follows:
goat anti-mouse IgG labeled with fluorescein isothiocyanate (FITC) or rhoda-
mine, goat anti-rat IgG labeled with rhodamine, and goat anti-guinea pig IgG
labeled with FITC. Antibodies were used at the following dilutions: 1:500, anti-
gB; 1:100, anti-CMV IE1/2; 1:50, anti-integrin ␣5; 1:50, anti-integrin ␣1; and
1:20, anti-HLA-G.
Immunochemistry. Samples were processed for double indirect immunofluo-
rescence localization as described previously (11, 15, 20). Briefly, the explants
and filters were rinsed in phosphate-buffered saline, fixed in 3% paraformalde-
hyde overnight, and infiltrated with 5 to 15% sucrose followed by embedding in
optimal-cutting-temperature compound. Before the final embedding step, the
explants and Matrigel were removed from the inserts; after embedding was
completed, they were frozen in liquid nitrogen. Sections (5 to 7 m) were cut on
a Hacker-Slee cryostat and collected on slides. Isolated cytotrophoblasts plated
on Matrigel-coated filters were fixed in 3% paraformaldehyde for 20 min,
washed, and permeabilized for 5 min with cold methanol. In some experiments
fixed tissue sections or cells were stained for 1 h with a mixture of rat anti-human
cytokeratin (to identify trophoblasts) and anti-CMV antibodies. In other exper-
iments the mixture contained anti-gB and an antibody that recognized either an
integrin or HLA-G. The samples were then washed and incubated with the
appropriate secondary antibodies conjugated to FITC or rhodamine. Samples
were viewed with a Zeiss Axiophot epifluorescence microscope equipped with
filters to selectively view the rhodamine and fluorescein images.
Invasion assay. Cytotrophoblast invasiveness was quantified in an in vitro
invasion assay (diagrammed in Fig. 8) as previously described (40). Briefly, the
cells were isolated and plated on Matrigel-coated filters (six total per experi-
ment), and half of the cultures were infected with CMV as described above.
After 48 h, the filter inserts, together with the cultured cells, were excised with a
scalpel blade. The samples were stained with a mixture of antibodies that rec-
ognized cytokeratin (7D3) and CMV IE1/2 proteins (CH160-5). Afterwards the
filters were mounted on slides. CMV infection was evaluated by assessing IE1/2
and cytokeratin expression on the top surface of the filter. Invasion was quanti-
fied by counting cytokeratin-positive cell processes that penetrated the Matrigel
and appeared on the underside of the filter. The entire experiment was repeated
three times.
RESULTS
CMV proteins are expressed in distinct patterns in placen-
tal cells in chorionic villi after infection either in vitro or in
utero. First, we investigated CMV infection of chorionic villi in
vitro using the culture model illustrated in Fig. 2A. As de-
scribed in Materials and Methods, an important part of the
experimental design was to show that the placentas from which
the chorionic villi were dissected had not been infected in
utero. Figure 3 shows tissue sections of villous explants that
were incubated for 4 days after infection with CMV. The sec-
tions were double stained with anticytokeratin to identify tro-
phoblast cells (Fig. 3A and C) and an MAb to CMV IE1/2
proteins to identify infected cells (Fig. 3B and D). Routinely,
syncytiotrophoblasts that cover the villous surface were not
infected and failed to stain with the MAb to CMV IE1/2
proteins. Unexpectedly, we observed nuclear staining of iso-
lated clusters of underlying cytotrophoblast stem cells (Fig.
3B). The pattern was distinctive; in each section, groups of ⱕ10
adjacent cells reacted with the antibody. We observed this
staining pattern in villous explants from seven different placen-
tas that were infected with CMV in vitro. In some explants
CMV IE1/2 protein expression was also detected in cytotro-
phoblasts found in the cell columns of anchoring villi (Fig. 3D).
In one instance, we found that the majority of cytotrophoblast
stem cells expressed CMV IE1/2 proteins (data not shown).
Explants from five other healthy placentas failed to develop
infection at 5 days after culture with CMV.
Because we were screening placentas for CMV IE1/2 pro-
tein expression prior to culture, we obtained five specimens
that had already been infected in utero. The staining patterns
that we saw had remarkable similarities to and differences from
those we observed after CMV infection in vitro. With regard to
similarities, in three specimens we observed areas in which
CMV IE1/2 protein staining patterns were virtually indistin-
guishable from those observed after infection in vitro; isolated
clusters of cytotrophoblasts underlying the syncytium were the
only CMV-infected cells (Fig. 4B). In one placenta, we found
that nearly all the cytotrophoblast stem cells (Fig. 4C), as well
as those found within columns, expressed CMV IE1/2 proteins
(Fig. 4D). Comparatively fewer syncytial nuclei stained, but
numerous cells within the villous cores expressed these CMV
proteins (Fig. 4C). Based on morphological criteria, these in-
cluded fibroblasts, macrophages, and endothelial cells. In a
different placenta we detected yet another staining pattern.
Nearly all the fibroblasts in the villous core expressed CMV
antigens. Comparatively fewer cytotrophoblasts were stained,
primarily clusters of villous stem cells. About 50% of syncy-
tiotrophoblasts were also stained.
CMV replicates and virions are released from differentiat-
ing cytotrophoblasts infected in vitro. We further investigated
CMV replication in human placental cells by using a second in
vitro model (illustrated in Fig. 2B). In this model, cytotropho-
blast stem cells isolated from chorionic villi are plated as a
monolayer on Matrigel. Under these culture conditions, the
cells form aggregates, analogous to columns, and differentiate
along the invasive pathway.
In these experiments cytotrophoblasts were plated and then
infected with CMV. Replication was assessed by immunolocal-
izing CMV IE1/2 proteins and a virion envelope glycoprotein,
gB, either immediately after isolation (control) or at various
intervals postinfection (experimental). IE1/2 protein expres-
sion was detected, in a nuclear pattern, from 24 h onward (data
not shown). From 72 h onward, staining for both IE1/2 (nu-
clear) and gB (cytoplasmic) proteins was detected (Fig. 5B). At
96 h postinfection, the accumulation of gB in cytoplasmic ves-
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icles was particularly striking (Fig. 5D). In 10 separate exper-
iments, 20 to 40% of the cells showed the latter staining pat-
tern at the end of the culture period.
Next, we compared titers of infectious progeny made in
CMV-infected cytotrophoblasts and HFF during a 6-day pe-
riod. To do so, we monitored virus levels in the intracellular
and extracellular compartments daily (Fig. 6). Cytotropho-
blasts were plated at fivefold-higher numbers than HFF to
account for the fact that cells in the middle of aggregates are
sequestered from virus (see Fig. 2B). For the same reason,
cytotrophoblasts were infected with a 10-fold-higher virus titer
than was used to infect HFF. Although yields were higher in
HFF, the placental cytotrophoblasts produced and released
into the medium substantial amounts of virus. The results of
FIG. 3. Cytotrophoblasts (CTBs) in villous explants were infected with CMV in vitro. Tissue sections prepared from both floating villi (FV; A and B) and anchoring
villi (AV; C and D) were analyzed by double staining with anticytokeratin and anti-CMV IE1/2. (A) Cytokeratin (CK) staining of floating villi showed the multinucleate
syncytiotrophoblasts (ST; arrowheads) that cover the villous surface and the underlying villous cytotrophoblast stem cells (CTBv; arrows). (B) CMV IE1/2 proteins were
expressed by underlying clusters of infected villous cytotrophoblast stem cells. The inner stromal villous cores (VC) were consistently negative for anti-IE1/2 antibody
staining. (C and D) IE1/2 protein expression was also detected in cytotrophoblasts found in the cell columns (CC) of anchoring villi. Insets show infected
cytotrophoblasts at higher magnification. As a control, staining was performed as described for the experimental situation except that the primary or secondary antibody
was omitted. No staining was detected (data not shown).
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FIG. 4. Cytotrophoblasts (CTBs) and other cells show evidence of natural infection of chorionic villi with CMV in utero. Both floating villi (FV; A to C) and
anchoring villi (AV; D) were studied. Tissues were analyzed by using immunolocalization techniques for expression of (A) cytokeratin (CK) and (B to D) CMV IE1/2
proteins. (B) In some cases, clusters of CMV-infected villous cytotrophoblast stem cells (CTBv; arrows) underlying the syncytium (ST, arrowheads) were the only sites
of antibody reactivity. More often, numerous cells throughout the villi stained with anti-IE1/2 antibody. (C) In floating villi, nuclei of syncytiotrophoblasts, villous
cytotrophoblasts, and stromal components expressed IE1/2 proteins. (D) The same pattern of immunoreactivity was seen in infected anchoring villi. Additionally,
cytotrophoblasts in cell columns (CC) stained brightly. As a control, staining was performed as described for the experimental situation except that the primary or
secondary antibody was omitted. No staining was detected (data not shown).
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these experiments indicated that differentiating or invading
cytotrophoblasts were fully permissive for CMV replication.
CMV infection in vitro downregulates ␣11 integrin expres-
sion and impairs cytotrophoblast invasion. The association of
CMV infection with pregnancy complications thought to in-
volve the placenta prompted us to examine the effects of CMV
infection on the expression of the laminin/collagen receptor
integrin ␣11. We studied this extracellular matrix receptor
both as a stage-specific antigen whose expression is preferen-
tially associated with cytotrophoblasts inside the uterine wall
(11) and as an adhesion molecule that mediates invasion in
vitro (12). First, we colocalized CMV gB (Fig. 7A and C) and
integrin ␣11 expression (Fig. 7B and D) in cytotrophoblast
cultures that were infected with CMV for 96 h in vitro. As
expected, the cells that did not stain for gB (Fig. 7A) expressed
integrin ␣1 in a plasma membrane pattern (Fig. 7B). Diffuse
cytoplasmic staining for gB was also correlated with integrin ␣1
expression (see cell marked with a * in C and D), but accu-
mulation of gB in vesicles (Fig. 7C) was associated with the
absence of staining for integrin ␣1 (Fig. 7D). In contrast, im-
munostaining for another integrin whose expression is upregu-
lated as the cells invade, the fibronectin receptor ␣51, was not
affected (data not shown). In this context it is interesting to
consider that ␣51 functions to inhibit invasion, thereby coun-
terbalancing the activity of integrin ␣11 (12).
Next, we evaluated the impact of CMV infection in vitro on
cytotrophoblast invasion, using the assay illustrated in Fig. 8.
Isolated cytotrophoblasts were plated on the upper surfaces of
Transwell filters coated with Matrigel to an approximate depth
of 100 m. The assay tests a cell’s ability to penetrate the
Matrigel, pass through pores in the underlying filter, and
emerge on the lower surface of the membrane (12, 40). Inva-
FIG. 5. Purified cytotrophoblasts (CTBs) could be infected with CMV as they differentiated along the invasive pathway in vitro. At 72 h, the cells were stained for
expression of (A) cytokeratin (CK) and (B) gB (the major structural glycoprotein in the virion envelope) and IE1/2 proteins. Anti-IE1/2 antibody reacted with the nuclei,
and anti-gB antibody showed diffuse cytoplasmic staining. At 96 h, the cells were stained for expression of (C) cytokeratin and (D) gB, which was detected in granules
in the cytoplasm.
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sion is quantified by determining the number of cytokeratin-
positive cell processes that emerge through the filter pores. In
three separate experiments we found that CMV infection in
vitro dramatically impaired invasion, which was reduced to
16% ⫾ 3.2% (mean ⫾ standard error of the mean) of that
observed in control uninfected cells. We noted that the effect
on invasion was greater than could be accounted for in terms
of the number of CMV-infected cells (e.g., 20 to 40%). This
result suggests that the presence of infected cells in the invad-
ing aggregates (see Fig. 2B) influences the behavior of the
population as a whole.
Figures 8A to D are micrographs showing typical filters. Two
days postinfection, many of the cytokeratin-positive cytotro-
phoblasts in the upper chamber (Fig. 8A) had become infected
with CMV in vitro, as shown by immunolocalization of CMV
IE1/2 proteins to the nuclei (Fig. 8B). This was in contrast to
control cytotrophoblasts maintained under the same culture
conditions in the absence of virus, which failed to demonstrate
any immunoreactivity (data not shown). Examination of the
filter underside from control cultures showed that most of the
pores contained cytokeratin-positive processes of cytotropho-
blasts that were emerging on the filter underside (Fig. 8C). In
contrast, the processes of CMV-infected cells showed diffuse
immunofluorescence because they had not yet penetrated the
Matrigel to reach the filter pores (Fig. 8D).
CMV infection in vitro downregulates cytotrophoblast ex-
pression of the nonclassical MHC class Ib molecule HLA-G.
Multiple loci in the CMV genome downregulate expression of
MHC class Ia molecules from the surface of infected cells (32).
Thus, we investigated whether CMV infection in vitro affects
FIG. 6. Purified cytotrophoblasts (CTBs) were fully permissive for CMV
infection in vitro. Quantitation and comparison of CMV progeny virions pro-
duced in cell extracts (intracellular virus) and culture medium (released virions)
of purified cytotrophoblasts and human foreskin fibroblasts (HFF) infected in
vitro. TCID
50
, 50% tissue culture infective dose.
FIG. 7. CMV infection in vitro eventually downregulates cytotrophoblast (CTB) expression of integrin ␣1. Purified cytotrophoblasts were infected with CMV in
vitro as described in Materials and Methods. At 72 h after infection, the cells were fixed and stained for expression of gB and integrin ␣1. Cytotrophoblasts that did
not express gB (A) displayed prominent staining for integrin ␣1 in a plasma membrane-associated pattern (B). Likewise, cells that stained in a diffuse cytoplasmic
pattern for gB (C) also reacted with the anti-integrin antibody (D, cell marked with *). However, when gB was localized in a vesicular pattern, integrin staining was
not detected (D). The intense intracellular staining was bleedthrough from anti-gB signal.
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expression of the cytotrophoblast MHC class Ib molecule
HLA-G. Immunolocalization experiments showed that at late
times after infection, when high levels of CMV gB were de-
tected (Fig. 9A), staining for HLA-G was either greatly re-
duced or lost (Fig. 9B). This was in contrast to cells in the same
microscope field (e.g., internal controls) that were not infected
with CMV and that stained with anti-HLA-G. To identify the
relevant CMV glycoproteins responsible, we infected cytotro-
phoblasts with two CMV mutants, RV798 and RV670, in
which all of the genes known to downregulate cell surface
expression of classical MHC class Ia molecules have been
deleted (33). The results of five separate experiments showed
that both mutants RV798 and RV670 downregulated HLA-G
expression in infected cytotrophoblasts from different placen-
FIG. 8. CMV infection impairs cytotrophoblast invasion in vitro. (Upper panel) Diagram of the assay that assesses the ability of cells to penetrate the Matrigel
substrate, migrate through pores in the underlying filter, and emerge on the underside. Purified cytotrophoblasts (CTB) were cultured on Matrigel-coated filters and
infected with CMV 12 h later. Staining of the upper surface of the filter (I) for (A) cytokeratin (CK) and (B) IE1/2 proteins expression showed that ⬃30% of the cells
were infected 48 h after plating. Invasion was quantified by determining the number of cytokeratin-positive cell processes that penetrated the Matrigel and appeared
in the pores (marked with arrowheads) that open on the underside of the filter (II). In control cultures (C) many processes were visible, whereas in CMV-infected
samples (D) only the pores were visible, indicating a significant reduction in invasion.
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tas (data not shown). Therefore, the mechanism of HLA-G
downregulation does not involve glycoproteins that alter class
Ia expression and is most likely novel.
DISCUSSION
The impetus for this study was the long-standing hypothesis
that CMV infection of the placenta precedes that of the em-
bryo or fetus, suggesting that the extraembryonic membranes
play a critical role in pathogenesis. Given the difficulties inher-
ent in studying the infection process during human pregnancy,
much of the direct experimental evidence in support of this
hypothesis comes from animal models. In this context it is
important to consider the tremendous diversity in placental
structure among animals—even close genetic relatives. For this
reason studies in the guinea pig, which, like the human, has a
hemomonochorial placenta in which a single trophoblast layer
separates the fetal from the maternal circulation, are of par-
ticular interest (23, 39). Dams inoculated in the axilla with
guinea pig CMV at midgestation show hematogenous dissem-
ination of infection to the placenta, where viral nucleocapsids
are present in nuclei of syncytiotrophoblasts and viral proteins
are expressed in the transitional zone between the capillarized
trophoblast labyrinth and the noncapillarized interlobium (23).
Furthermore, CMV, which replicates in the presence of ma-
ternal antiviral antibodies, is detected in placental tissues long
after virus is cleared from blood. Whenever infection of the
fetus occurred, virus was isolated from the associated placenta.
Conversely, when CMV infected the placentas, only 27% of
fetuses contained virus, suggesting that the guinea pig placenta
serves as a reservoir in which virus replicates prior to reaching
the embryo or fetus.
Our results suggest that this is also the likely scenario in
human pregnancy and that CMV-infected cytotrophoblasts
play a central role in virus transmission to the fetus. We found
evidence of CMV replication in the trophoblast populations
that lie at the maternal-fetal interface, either in vitro or in
utero. Specifically, trophoblast cells in several locations ex-
pressed CMV proteins after infection. Given the 3- to 4-day
CMV replication cycle, the in vitro studies, in which we de-
tected infection of cytotrophoblast stem cells as well as of the
invasive subpopulation, likely model the initial steps in virus
transmission. In contrast, the tissues infected in utero show
how the virus is transmitted from trophoblasts to other types of
cells within the villous core.
These findings offer important clues about how transmission
occurs in utero. In reconstructing possible routes, we consid-
ered immunohistochemical analyses of CMV-infected placen-
tas (44, 45, 55) and recent data showing that CMV persistently
or latently infects many of the cell types that trophoblasts
encounter in the uterus. Specifically, CMV establishes latent
infection in and reactivates from granulocyte-dendritic progen-
itors (25, 56). Macrophages disseminate virus by contact with
endothelial cells that line blood vessels and tissues of solid
organs (63). CMV also directly infects endothelial cells in vivo,
which have subsequently been found circulating in blood (22).
Uterine tissues may become infected via a hematogenous route
or by sexual contact; currently there is no evidence that
strongly supports either mechanism. Circumstantial evidence
for sexual transmission includes high rates of CMV infection in
sexually active adolescents who are likely to become pregnant
(7, 57). Consequently, CMV infection would spread in an as-
cending manner from the cervix to the uterus in cases of
primary or reactivated infections. In support of this hypothesis,
CMV is shed from the cervix in young nonpregnant women
with multiple new sex partners (5–7, 57), and this rate increases
during pregnancy (35%) (4, 60). High levels of CMV DNA are
detected in cervical smears and uterine tissues (50% positive)
compared with lung, liver, kidney, and blood vessels (15%
positive), and viral proteins are detected in uterine glandular
epithelial cells, endothelial cells, and interstitial leukocytes
(19). Together, these data indicate that CMV productively
replicates in and is shed from uterine tissues of sexually active
women.
These data also suggest possible routes by which CMV in-
fection spreads from the uterine tissues, first to the placenta
and then to the embryo or fetus. One likely site of transmission
is via the syncytiotrophoblast layer that covers floating chori-
onic villi (Fig. 1, site 1). These placental cells are also in direct
contact with maternal blood. Our data suggest that initially the
syncytium may function by allowing passage of CMV to the
underlying layer of cytotrophoblast stem cells, which are capa-
ble of supporting viral replication. Later in the infection pro-
cess syncytiotrophoblasts may also become infected. Another
FIG. 9. CMV infection impairs cytotrophoblast expression of HLA-G in
vitro. Purified cytotrophoblasts were isolated and infected with CMV as de-
scribed in Materials and Methods. At 72 h after infection, the cells were stained
for gB (A) and HLA-G (B) expression. Cells that did not express gB (black
arrows) expressed HLA-G. In contrast, staining for gB (white arrows) was asso-
ciated with a marked reduction in HLA-G expression.
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likely site of transmission is within the uterine wall (Fig. 1, sites
2 and 3). Cytotrophoblasts involved in interstitial invasion
could encounter infected uterine glands, decidual granular leu-
kocytes, and muscle cells. Cytotrophoblasts involved in endo-
vascular invasion could encounter infected endothelial and
vascular smooth muscle cells as well as maternal blood. Once
cytotrophoblasts within the uterine wall become infected,
CMV could spread in a retrograde manner through the cell
columns to the anchoring chorionic villi. We also saw, in a
number of samples infected in utero, extensive expression of
CMV IE1/2 proteins throughout the villous stromal cores. This
unexpected result suggests that virus is often transmitted from
infected trophoblasts to fibroblasts, fetal macrophages (Hof-
bauer cells), and possibly endothelial cells that line chorionic
vessels—patterns of CMV infection in the placenta and other
tissues (45, 54). Infected macrophages and sloughed endothe-
lial cells seem likely candidates for entering the venous circu-
lation of the placenta and subsequently carrying the infection
via the placental circulation to the fetus.
It is equally interesting to consider the possible molecular
cascade that results in CMV transmission via the placenta to
the fetus. With regard to transmission within the uterine wall,
the best analogy may be reactivation of CMV in transplant
patients whose immune systems have been pharmacologically
suppressed. Likewise, the placenta, which is often described as
a hemiallograft, probably induces a state of local immunosup-
pression in the uterus. For example, the invasive cytotropho-
blast subpopulation secretes high levels of interleukin-10 (50).
We speculate that this specialized immunologic milieu could
support reactivation of latent virus. With regard to transmis-
sion in the intervillous space, several possible mechanisms ex-
ist. For example, human syncytiotrophoblasts express the neo-
natal Fc receptor hFcRn, which transcytoses IgG from
maternal blood to the fetus (53). The abundance of nonneu-
tralizing antiviral antibodies with low avidity in women with
primary CMV infection who transmit virus to the embryo or
fetus (2, 37) may enhance virion transcytosis across syncy-
tiotrophoblasts to cytotrophoblast stem cells. This finding is in
accord with our observation that, in floating villi infected with
CMV in vitro, syncytiotrophoblasts failed to stain with anti-
bodies to viral proteins, whereas clusters of underlying cytotro-
phoblasts did. Currently we are investigating whether hFcRn
expressed at the apical surface of syncytiotrophoblasts binds
and transports both maternal IgG, which we and others (36,
53) have localized to submembrane vesicles in these cells, and
antibody-coated CMV virions. Interestingly, virus transmission
to the embryo or fetus by transcytosis in syncytiotrophoblasts
would explain the phenomenon of efficient intrauterine infec-
tion in the presence of high antibody titers to CMV gB (2).
Others have reported that CMV replicates in trophoblasts
from first-trimester and full-term placentas infected in vitro
(26, 29). CMV virions were found in trophoblast culture me-
dium, and infection kinetics varied among laboratory strains
and virus isolates. These studies did not address the conse-
quences of infection on cytotrophoblast function. Our data
suggest that CMV impairs cytotrophoblast differentiation/in-
vasion in vitro. Experiments currently in progress are address-
ing the critical question of whether these same changes are
seen as a consequence of infection in utero. Data gathered thus
far suggest that this is the case. To date we have isolated
cytotrophoblasts from two second-trimester placentas that had
been naturally infected with CMV in utero, as demonstrated by
their nuclear expression of IE1/2 proteins and cytoplasmic
expression of gB before culture. After 3 days, they continued to
express gB and expression of both ␣11 integrin and HLA-G
was downregulated. In both cases invasiveness after 2 days in
culture, quantified by using the assay depicted in Fig. 8, was
only ⬃5% of the levels commonly observed in cultures of
gestation-matched uninfected cells.
Therefore, it seems likely that the extensive infection of the
trophoblast populations that we detected in first-trimester cho-
rionic villi infected in utero could adversely affect placental
development and consequently the outcome of the pregnancy.
The downstream consequences are likely to vary depending on
the gestational age. Infection of trophoblasts soon after im-
plantation might compromise the ability of the human embryo
to carry out interstitial implantation, which buries the concep-
tus deep within the uterine wall. This could explain the early
pregnancy loss that often occurs in women with primary infec-
tion. Infection at a slightly later stage could impair the forma-
tion of both floating and anchoring villi. In the former case,
placental structure may remain relatively undeveloped, per-
haps exhibiting the reduction in surface area of the villous tree
that has been noted in intrauterine growth retardation. In the
latter case, a constellation of critical events could be affected,
including the attachment of cell columns to the uterus and
both interstitial and endovascular invasion—placental pathol-
ogies that are associated with preeclampsia and a subset of
pregnancies that are complicated by idiopathic preterm labor
(49). Although the consequences of CMV infection of the
developing trophoblast in early pregnancy are not known, the
effects that we propose could explain why CMV infection later
in pregnancy is frequently associated with both intrauterine
growth retardation and preterm labor (31).
Our results also indicate that CMV infection impairs cy-
totrophoblast expression of HLA-G, likely an important com-
ponent of the mechanism that protects these fetal cells from
removal by maternal immune cells that are abundant in the
decidua, particularly during the first trimester of pregnancy.
This is in accord with the previously reported effects of CMV
infection on HLA-G expression by human choriocarcinoma
cells (52). Furthermore, the mechanism of HLA-G downregu-
lation does not involve CMV genes that alter class Ia expres-
sion and is most likely novel. This finding is in accord with the
fact that expression of HLA-G, which lacks an interferon re-
sponse element in its promoter, is regulated in a manner dis-
tinct from that of class Ia molecules (8). One consequence of
downregulating HLA-G expression could be activation of the
maternal immune response against the subpopulation of cy-
totrophoblasts that express this molecule—namely, those that
carry out interstitial and endovascular invasion. Thus, it is
possible that infected cytotrophoblasts become targets of the
unusual natural killer (NK) cell population that dominates the
granular leukocyte population in the uterine decidua (61). As
noted above, the timing of infection would determine the effect
on pregnancy outcome.
The data presented in this study also raise several interesting
questions that we cannot yet answer. For example, it is possi-
ble, even probable, that the widespread expression of CMV
IE1/2 proteins in first-trimester chorionic villi that were in-
fected in utero could be evidence of other underlying pathol-
ogies, including those involving infectious organisms other
than CMV. Given the complex interplay between viruses, bac-
teria, and host cells that takes place in the uterine environ-
ment, this scenario seems very likely (21). Thus, it will be
important to place our findings in the larger context of the
microbial ecology of the female reproductive tissues. Finally,
CMV infection may be indicative of abnormal cross talk be-
tween the fetal and maternal cells that orchestrate the complex
immune interactions required for human pregnancy to proceed
normally. Imbalances in trophoblast differentiation, decidual-
6818 FISHER ET AL. J. VIROL.
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ization, and/or decidual granular leukocyte infiltration could
be related to the phenomena that we observed.
In summary, our findings open the door to testing a variety
of hypotheses regarding CMV infection of placental tissues. It
is hoped that these studies will resolve the serious dichotomy
between our understanding of the devastating consequences of
congenital CMV infection and our lack of knowledge, at the
molecular level, of the mechanisms involved. Understanding
how CMV transmission occurs is the crucial first step toward
the rational design of therapies to prevent prenatal infection.
These treatments could either enhance the normal barrier
function of the placenta or subvert the ability of maternal cells
to transmit CMV to cytotrophoblasts—fetal placental cells that
are the likely conduit for CMV infection of the embryo or fetus.
ACKNOWLEDGMENTS
All authors contributed equally to this paper.
We thank Thomas Jones for CMV deletion mutant viruses. We also
thank Edward Mocarski and members of the Fisher and Pereira labs
for thoughtful discussions. We are grateful to Zoya Kharitonov for
excellent laboratory expertise and Evangeline Leash for editing the
manuscript.
This work was supported by Public Health Service grants HD30367
(S.F.), EY10138 (L.P.), and AI46657 (L.P. and S.F.) from the National
Institutes of Health.
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