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Journal of Virology, September 2006, p. 8970-8980, Vol. 80, No. 18
0022-538X/06/$08.00+0     doi:10.1128/JVI.00296-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Role for 3-O-Sulfated Heparan Sulfate as the Receptor for Herpes Simplex Virus Type 1 Entry into Primary Human Corneal Fibroblasts

Vaibhav Tiwari,1,{dagger} Christian Clement,1,2,{dagger} Ding Xu,3 Tibor Valyi-Nagy,4 Beatrice Y. J. T. Yue,1 Jian Liu,3 and Deepak Shukla1,2*

Department of Ophthalmology and Visual Sciences,1 Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612,2 Division of Medicinal Chemistry and Natural Products, University of North Carolina, Chapel Hill, North Carolina 27599,3 Department of Pathology College of Medicine, University of Illinois at Chicago, Chicago, Illinois4

Received 9 February 2006/ Accepted 19 June 2006

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.

* Corresponding author. Mailing address: University of Illinois at Chicago, Lions of Illinois Eye Research Institute, M/C 648, 1855 West Taylor Street, Chicago, IL 60612. Phone: (312) 355-0908. Fax: (312) 996-7773. E-mail: dshukla{at}uic.edu.

{dagger} V.T. and C.C. contributed equally to this work.

Journal of Virology, September 2006, p. 8970-8980, Vol. 80, No. 18
0022-538X/06/$08.00+0     doi:10.1128/JVI.00296-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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