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Abstract
Human cytomegalovirus (HCMV) is the leading viral cause of congenital malformation. Neonates with HCMV infection can develop a wide range of symptoms at birth, as well as permanent neurologic sequelae and, in severe cases, intrauterine and postnatal death. To transmit vertically from the mother, HCMV must traverse the placental barrier, a complex multicellular tissue. Three HCMV genes, UL128, UL130 and UL131A, were previously shown to be essential for cellular entry in epithelial and endothelial cells, as well as cell-to-cell spread and syncytia formation. Within this investigation, RNA interference (RNAi) was used to silence the HCMV UL130 locus and analyse the effect upon viral replication and spread. Three siRNA molecules siUL130A, siUL130B and siUL130C were designed to initiate and direct RNAi-mediated silencing of the UL130 locus in epithelial (ARPE-19), trophoblast (TEV-1) and fibroblast (MRC-5) cell lines. Merlin, a clinical strain of HCMV, was used to infect these cell lines. Viral titre was analysed on day 1, 4, 7 and 10 post-transfection. Effects upon cell-to-cell spread were examined using the immunofluorescent staining of three prominent HCMV proteins: IE, pp65 and gB. Two of the siRNA molecules, siUL130A and siUL130B, induced a reduction in viral replication and spread in epithelial cells and trophoblasts when compared to a scrambled siRNA control. In fibroblasts, however, these molecules increased viral replication but had no effect upon viral spread. The third siRNA molecule, siUL130C, was not effective; it induced an initial increase in viral replication and had no effect upon viral spread. These results highlight the importance of the UL130 locus in placental infection and transmission. The differential effect of UL130 knock down in fibroblasts and epithelia/trophoblasts indicates a potential role for siRNAs targeting UL130 in tissue specific treatment of HCMV infection. siRNAs, in contrast to the current antiviral treatments, have the potential to provide an alternative, highly specific and less toxic therapeutic for the prevention of HCMV infection and disease.