Developmental Regulation of Human Cytomegalovirus Receptors in Cytotrophoblasts Correlates with Distinct Replication Sites in the Placenta

Abstract

Cytomegalovirus (CMV), the major viral cause of congenital disease, infects the uterus and developing placenta and spreads to the fetus throughout gestation. Virus replicates in invasive cytotrophoblasts in the decidua, and maternal immunoglobulin G (IgG)-CMV virion complexes, which are transcytosed by the neonatal Fc receptor across syncytiotrophoblasts, infect underlying cytotrophoblasts in chorionic villi. Immunity is central to protection of the placenta-fetal unit: infection can occur when IgG has a low neutralizing titer. Here we used immunohistochemical and function-blocking methods to correlate infection in the placenta with expression of potential CMV receptors in situ and in vitro. In placental villi, syncytiotrophoblasts express the virion receptor epidermal growth factor receptor (EGFR) but lack integrin coreceptors, and virion uptake occurs without replication. Focal infection can occur when transcytosed virions reach EGFR-expressing cytotrophoblasts that selectively initiate expression of ␣V integrin. In cell columns, proximal cytotrophoblasts lack receptors and distal cells express integrins ␣1␤1 and ␣V␤3, enabling virion attachment. In the decidua, invasive cytotrophoblasts expressing coreceptors upregulate EGFR, thereby dramatically increasing susceptibility to infection. Our findings indicate that virion interactions with cytotrophoblasts expressing receptors in the placenta (i) change as the cells differentiate and (ii) correlate with spatially distinct sites of CMV replication in maternal and fetal compartments.Human cytomegalovirus (CMV) is the leading cause of congenital viral infection in children, with an incidence in the United States of about 1 to 3% of live births. Primary CMV infection during gestation poses a 40 to 50% risk of intrauterine transmission (5), whereas reactivated infection in seropositive women rarely causes symptomatic disease, highlighting the role of immunity in fetal protection (16). Symptomatic infants have intrauterine growth restriction, and most survivors (28%) have permanent sequelae, including neurological defects, mental retardation, retinopathy, and sensorineuronal deafness (12). Although virus transmission can occur throughout pregnancy, congenital disease is more severe when primary infection takes place during early gestation (54). Intrauterine growth restriction and loss of the fetus without virus transmission, which are associated with congenital CMV infection, originate in placental pathology (3,21).Placentation is a stepwise process whereby specialized cytotrophoblast progenitor cells leave the basement membrane to initiate blood flow, differentiating along two pathways depending on their location (Fig. 1). In floating villi, cells fuse to form a multinucleate syncytial covering attached at one end to the tree-like fetal portion of the placenta. Covered by syncytiotrophoblasts, these villi float in a stream of maternal blood, a source of nutrients and immunoglobulin G (IgG) transported to the fetus. In anchoring villi, cytotrophoblasts switch from an ...

Activation of EGFR on monocytes is required for human cytomegalovirus entry and mediates cellular motility

“…qRT-PCR analysis confirmed EGFR-mediated viral entry (Fig. 2G) and revealed a 41% and 59% decrease in viral entry in the presence of neutralizing-EGFR antibody and AG1478, respectively, which is consistent with the 63% reduction in infectivity observed in trophoblasts treated with an anti-EGFR antibody before HCMV infection (11). Other growth factor-like receptor tyrosine kinases, including PDGFR-␣, VEGF receptor 1 (VEGFR1), and VEGFR2, are not involved in mediating viral entry into monocytes (Fig.…”

“…Several studies have reported the rapid activation of EGFR to occur in various cell lines following HCMV infection (8)(9)(10) and that the activation of EGFR signaling was required for efficient infection (8,11). We similarly found that EGFR activity was necessary for HCMV entry into primary monocytes, although in contrast to model cell types (23), activation of the downstream target PI(3)K was not required for HCMV entry.…”

“…19), it is unlikely that these receptors direct HCMV infection toward the monocyte subpopulation. EGFR has been shown to act as a viral tropism receptor for targeting HCMV entry into biologically distinct populations of trophoblast cells (11). Our data now provide evidence that EGFR expression and the ensuing downstream signaling following viral engagement on monocytes provide cell type specificity for viral internalization.…”

“…Following a temperature shift to 37°C to initiate internalization, we found TB40E in the cyto- plasm of the infected cell. Pretreatment of monocytes with AG1478 or a neutralizing EGFR antibody resulted in the inhibition of internalization of the viral capsid and the clustering of viral particles at the cell surface, which was also observed in EGFR-negative cytotrophoblasts (11). To assess the contribution of EGFR in the process of viral entry further, cells were examined for the presence of internalized HCMV genomic DNA.…”

“…However, unlike the transient activation observed in fibroblasts (8) and trophoblasts (10), HCMV infection stimulated chronic activation of EGFR in ECs (9), suggesting that distinct signaling profiles originating from the same cellular receptor can occur in different cell types. EGFR expression was required for viral entry/infection of breast cancer cells and trophoblasts (8,11). However, the role that EGFR plays during viral infection remains controversial, because it has been reported that EGFR does not mediate entry into fibroblasts (12) and others have shown that PDGF receptor (PDGFR)-␣ rather than EGFR was required for efficient viral entry (13).…”