Abstract
Abstract: :
Purpose: To investigate entry of herpes simplex virus (HSV) type-1 and –2 in human corneal fibroblasts and to identify the entry mediators. Methods: Entry assays were based on quantitation of ß-galactosidase expressed from the viral genome. Cultures of human corneal fibroblasts were derived from the stroma of donor eyes. Cells were plated in 96 well plates and after overnight incubation, were infected with recombinant ß-galactosidase expressing strains of HSV-1 (KOS) and HSV-2 (333). The entry of the HSV in corneal fibroblasts was quantitated by spectrophotometry. The role of viral glycoprotien gD and its cellular receptors, HVEM and Nectin-1, were examined by intereference and RT-PCR assays. Results: Our results clearly indicate that primary stromal cells are susceptible to entry of both HSV-1 and HSV-2. A comparative study with Nectin-1 expressing Chinese hamster ovary (CHO-K1) cells in parallel with the stromal cells showed that HSV-1 entry was more prominent in stromal cells than that of HSV-2. Cellular expression of viral glycoprotein gD rendered these cells resistant to HSV-1 entry. This gD-mediated interference explains an interaction between cell–associated gD and available gD-binding receptors. Furthermore, analysis by RT-PCR demonstrated the expression of only HVEM, and not Nectin-1. Therefore, it is likely that HSV entry in stromal cells is mediated by HVEM alone. Treatment of stromal cells with heparinase to remove cell surface heparan sulfate and with sodium chlorate, a potent inhibitor of proteoglycan sulfation, resulted into reduced HSV entry suggesting the importance of cell surface proteoglycans during initial binding or attachment of virus to the primary cells. Conclusions: Human corneal fibroblasts are permissive to HSV-1 and HSV-2 infections and widely express HVEM as the functional gD receptor for HSV entry. This is the first report demonstrating the entry mediators of pathogenic herpes simplex virus in corneal fibroblasts.
Keywords: herpes simplex virus • cornea: stroma and keratocytes • molecular biology