title: Role for 3-O-sulfated heparan sulfate as the receptor for herpes simplex virus type 1 entry into primary human corneal fibroblasts.
abstract: Herpes simplex virus type 1 (HSV-1) infection of the corneal stroma remains a major cause of blindness. Primary cultures of corneal fibroblasts (CF) were tested and found susceptible to HSV-1 entry, which was confirmed by deconvolution imaging of infected cells. Plaque assay and real-time PCR demonstrated viral replication and hence a productive infection of CF by HSV-1. A role for glycoprotein D (gD) receptors in cultured CF was determined by gD interference assay. Reverse transcription-PCR analysis indicated expression of herpesvirus entry mediator and 3-O-sulfated (3-OS) heparan sulfate (HS)-generating enzyme 3-O sulfotransferase 3 (3-OST-3) but not nectin-1 or nectin-2. Subsequently, HS isolated from these cells was found to contain two distinct disaccharides (IdoUA2S-AnMan3S and IdoUA2S-AnMan3S6S) that are representative of 3-OST-3 activity. The following lines of evidence supported the important role of 3-OS HS as the mediator of HSV-1 entry into CF. (i) Blockage of entry was observed in CF treated with heparinases. The same enzymes had significantly less effect on HeLa cells that use nectin-1 as the entry receptor. (ii) Enzymatic removal of cell surface HS also removed the major gD-binding receptor, as evident from the reduced binding of gD to cells. (iii) Spinoculation assay demonstrated that entry blockage by heparinase treatment included the membrane fusion step. (iv) HSV-1 glycoprotein-induced cell-to-cell fusion was inhibited by either prior treatment of cells with heparinases or by HS preparations enriched in 3-OS HS. Taken together, the data in this report provide novel information on the role of 3-OS HS in mediating infection of CF, a natural target cell type.
authors.affiliation: University of Illinois at Chicago, Lions of Illinois Eye Research Institute, M/C 648, 1855 West Taylor Street, Chicago, IL 60612, USA.
authors.forename: Vaibhav
authors.initials: V
authors.lastname: Tiwari
authors.forename: Christian
authors.initials: C
authors.lastname: Clement
authors.forename: Ding
authors.initials: D
authors.lastname: Xu
authors.forename: Tibor
authors.initials: T
authors.lastname: Valyi-Nagy
authors.forename: Beatrice Y J T
authors.initials: BY
authors.lastname: Yue
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
authors.forename: Deepak
authors.initials: D
authors.lastname: Shukla
mesh_terms: D000818:Animals; D016466:CHO Cells; D003315:Cornea / Q000166:cytology / Q000821:virology*; D006224:Cricetinae; D004187:Disaccharides / Q000737:chemistry; D005347:Fibroblasts / Q000166:cytology / Q000821:virology*; D006023:Glycoproteins / Q000378:metabolism; D006367:HeLa Cells; D019763:Heparin Lyase / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry*; D018259:Herpesvirus 1, Human / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding
chemical_list: D004187:Disaccharides; D006023:Glycoproteins; D019763:Heparin Lyase; D006497:Heparan Sulfate