Herpetic Stromal Keratitis: Virus Entry Into Corneal Fibroblasts Is Mediated by 3–O–Sulfated Heparan Sulfate

Abstract

Abstract: : Purpose: To identify the receptor used for Herpes Simplex Virus–1 entry into corneal fibroblasts cultured from the stroma of the human cornea. Methods: First, to verify the natural susceptibility of cultured corneal fibroblasts (CF) in vitro, the cells were exposed to ß–galactosidase–expressing and enhanced green fluorescent protein–expressing HSV–1 virions. Deconvolution and confocal microscopy were used to monitor entry into live cells. Immunohistochemistry and reverse transcriptase–PCR were used to examine entry receptors in CF. Disaccharide analysis of surface heparan sulfate isolated from CF was performed to verify the expression of 3–O–sulfated heparan sulfate. Spinoculation, antibody blocking and viral glycoprotein D (gD) binding assays were performed to verify receptor usage by CF. Results: A three dimensional model of HSV–1 entry into CF was generated by use of deconvolution and confocal microscopy. Expression of HSV–1 gD rendered resistance to HSV–1 entry suggesting an important role for gD receptors. Expression of HVEM and 3–O sulfated heparan sulfate (3–OS HS), but not nectin–1, was detected by reverse transcriptase–PCR. Absence of nectin–1 was also evident from immunohistochemistry and resistance of cultured CF to bovineherpesvirus–1, which uses nectin–1 for entry. Demonstrating the significance of HVEM in entry, anti–HVEM antibody partially blocked HSV–1 entry. This effect was more pronounced when combined with heparinase treatment; suggesting an important role for HS. Finally, using a spinoculation technique, it was found that heparinase treatment of cultured CF blocked entry at fusion step, implicating 3–OS HS as a mediator of membrane fusion. Presence of 3–OS HS on CF was also verified by disaccharide analysis with its gD binding ability established by an immunoprecipitation assay. Involvement of an alternate receptor other than HVEM or nectin–1 was also suggested by blocking of HSV–1 entry by an anti–gD monoclonal antibody DL6, which blocks entry mediated by 3–OS HS but not via nectin–1 or HVEM. Conclusions: We provide molecular and biochemical evidence that HSV–1 entry into primary cultures of human corneal fibroblasts is mediated by HVEM and 3–OS HS but not nectin–1.

Keywords: herpes simplex virus • keratitis • cornea: stroma and keratocytes