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Abstract
Human cytomegalovirus (CMV) is the major infectious cause of congenital malformation in developed countries. CMV is transmitted from the pregnant mother to the fetus via the placenta, with placental trophoblasts being recognised as an important reservoir for the virus. CMV infection can also lead to changes in cytokine profiles, and inflammation of the placenta, with implications for pathogenesis of fetal disease. This study investigated the importance of viral pentameric complex of gH/gL/pUL128-pUL131A, and cellular platelet-derived growth factor receptor-α (PDGFRα) for CMV infection in placental trophoblasts. The relationship between CMV infection and cellular changes, as well as the immunomodulatory properties of CMV-encoded proteins were also examined.
Investigation in trophoblast cell lines has revealed CMV was able to enter these cells via different pathways, involving at least the viral pentameric complex of gH/gL/pUL128-pUL131A and cellular PDGFRα receptor. Infection with four CMV strains (Merlin, TB40E, Towne, AD169), and mutant Merlin strains with deletion of individual open reading frames UL128, UL130, or UL131A, showed the viral pentameric complex was essential for infection of HTR-8/SVneo trophoblasts, but non-essential for infection of SGHPL-4 trophoblasts. Analysis of the cell surface receptor PDGFRα, a receptor for CMV entry into human fibroblasts and glioma cells, showed differential expression of the receptor in these trophoblasts. Neutralisation, and transient expression of PDGFRα in respective SGHPL-4 and HTR-8/SVneo trophoblasts, demonstrated PDGFRα provides a different entry pathway for CMV that is independent of the viral gH/gL/pUL128-pUL131A pentameric complex.
CMV infection has been shown to induce many host genes, many of which encode for inflammatory proteins. Analysis of the pro-inflammatory cytokines (CCL2 and TNF-α) in CMV-infected fibroblasts showed significant alteration in intracellular and extracellular CCL2 levels during the course of infection. Comparison between live and UV-irradiated viral infections showed changes in CCL2 levels were a direct response to active CMV replication. There were no significant changes in TNF-α expression during a parallel time course of CMV infection. In transient transfection assays, overexpression of CMV tegument protein pp71 resulted in intracellular and extracellular upregulation of CCL2, which was mediated through transcriptional upregulation. Therefore, CMV-induced upregulation of CCL2 during early stages of infection is mediated, at least in part, by stimulation of viral pp71.
Infection with CMV clinical strain Merlin resulted in a pro-inflammatory shift in cytokine profiles in trophoblast cultures, suggesting infected trophoblasts are key contributors for production of inflammatory mediators within the placental tissue. Using the siRNA-targeted inhibition of UL111A and ORF94 gene transcripts, the immunomodulatory properties of these gene products were also investigated in infected SGHPL-4 trophoblasts. Notably, the viral replication in trophoblasts was reduced upon inhibition of UL111A and ORF94 transcripts, although this effect was not observed in MRC-5 fibroblasts. There was no evidence for modulation of cytokine levels in infected trophoblast cultures by UL111A and ORF94 gene products, suggesting these genes may not be critical for immune evasion during CMV infection of the placenta.
Despite advances in the diagnosis of maternal and fetal CMV infection, an effective therapy remains unavailable to prevent materno-fetal transmission of the virus, and the adverse pregnancy outcomes associated with infection. This study demonstrated that CMV uses multiple entry pathways, involving at least the viral pentameric complex of gH/gL/pUL128-pUL131A and PDGFRα receptor for entry into placental trophoblasts. This added to the existing knowledge of gB-mediated entry into trophoblast progenitor cells, possibly via co-expressed epidermal growth factor receptor and integrin receptors. These findings indicate therapeutic treatment targeting multiple entry pathways is required for efficient blocking of CMV infection in placental trophoblasts (and possibly other placental cell types), thereby preventing the transmission of virus across the placenta during pregnancy.
The induction of CCL2 by infected fibroblasts and trophoblasts indicates immune cells are potentially attracted to the sites of infection and mediates the inflammatory process within the placenta. On the other hand, uninfected immune cells attracted to the site of CMV infection may become infected, and facilitate dissemination of the virus. In addition to the induction of CCL2, CMV-infected placental trophoblasts can also elicit a strong inflammatory response through induction of many pro-inflammatory cytokines. The Th1 predominant pro-inflammatory shift in cytokine profiles has important consequences for placental development, normal placental function, materno-fetal virus transmission, fetal growth and fetal viability. Further investigations are required for improved understanding of virus-cell interactions, particularly, CMV entry and infection in different primary trophoblast cell types, as well as the immunomodulatory properties of other CMV gene products in these trophoblasts.