Replication in Differentiating Invading Cytotrophoblasts

Analysis of first- and second-trimester placentas from women with moderate CMV neutralizing titers showed that interstitial cytotrophoblasts in decidua are infected in utero (Pereira et al. 2003). As cytotrophoblasts differentiate and progress along

Fig. 4 CMV replicates in invasive cytotrophoblasts that express virion receptors. a Diagram of differentiating/invading cytotrophoblasts. The blue field represents the localization of viral proteins in b, c, d, e, and f. b Invasive cells in decidua infected in utero. The inset corresponds to the outlined area in the panel. Toledorec virions (green) bind to cytotrophoblast membranes with EGFR (c), integrin aipi (d), and integrin aV (e) (red) at 1 h after infection. f IE1 and IE2 proteins (green) in cytokeratin (CK)-stained cells (red) at 24 h after infection. Nuclei were counterstained with TO-PRO-3 iodide (blue)

Fig. 4 CMV replicates in invasive cytotrophoblasts that express virion receptors. a Diagram of differentiating/invading cytotrophoblasts. The blue field represents the localization of viral proteins in b, c, d, e, and f. b Invasive cells in decidua infected in utero. The inset corresponds to the outlined area in the panel. Toledorec virions (green) bind to cytotrophoblast membranes with EGFR (c), integrin aipi (d), and integrin aV (e) (red) at 1 h after infection. f IE1 and IE2 proteins (green) in cytokeratin (CK)-stained cells (red) at 24 h after infection. Nuclei were counterstained with TO-PRO-3 iodide (blue)

the invasive pathway, integrins aipi and aVp3 are upregulated (Damsky et al. 1994; Zhou et al. 1997). Location of CMV-infected invasive cytotrophoblasts is illustrated in Fig. 4a. Analysis of early gestation decidua from a placenta with low neutralizing titers showed that infected cytotrophoblasts express viral replication proteins (Fig. 4b). Cultures of differentiated cytotrophoblasts on filters coated with Matrigel were used to monitor these cells for expression of potential receptors, binding of Toledorec virions and productive infection. Virions (punctate green) adsorbed to membranes of cells expressing EGFR (Fig. 4c) and integrins aipi (Fig. 4d) and aV (Fig. 4e) at 1 h after infection. In addition, CMV IE1- and IE2-infected cell proteins were detected in nuclei of Toledo-infected cytotrophoblasts at 24 h, suggesting early-stage viral replication (Fig. 4f). These results showed that virion attachment and infection of invasive cytotrophoblasts occur in vitro in cells that express CMV receptors, EGFR and integrins aV and a1p1.

Using function-perturbing antibodies and soluble integrins, we evaluated the role of molecules that function as potential receptors in differentiating/invasive cytotrophoblasts (Maidji et al. 2007). Intense expression of EGFR and integrins a1p1 and aVp3 was found as cytotrophoblasts differentiated and invaded. Moreover, caveolin-i was expressed, as reported for syncytiotrophoblasts and villous cytotrophoblasts where CMV virion gB was localized (Maidji et al. 2006). Function-blocking antibody to EGFR blocked 63% of infectivity, whereas antibody to integrin pi blocked 80% and antibody to integrin aV blocked 43%. Function-blocking antibody to integrin a5 and isotype controls failed to reduce viral replication. When CMV virions were pretreated with soluble forms of integrins before infection, aipi blocked i00% of virion infectivity. Pretreatment with soluble integrin aVp3 reduced infectivity by approximately 37%, and pretreatment with soluble integrin a3pi reduced infectivity by i3%. Together the results of function-perturbing experiments confirmed that EGFR and integrins aipi and aVp3 - developmentally regulated molecules - function as CMV receptors as cytotrophoblasts progress along the differentiation pathway.

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