title: Spinoculation of heparan sulfate deficient cells enhances HSV-1 entry, but does not abolish the need for essential glycoproteins in viral fusion.
abstract: Cell surface heparan sulfate functions as a co-receptor in HSV-1 entry. In order to study its significance in context with specific gD receptors (nectin-1, HVEM, and 3-O-sulfated heparan sulfate) a low speed centrifugation based virus inoculation (spinoculation) method was used. The experiments were performed at 1200 x g using glycosylaminoglycan positive (GAG+) or deficient (GAG-) cells expressing gD receptors. Clearly, spinoculation of GAG- nectin-1 or HVEM cells enhanced significantly viral entry compared to similar but unspun cells. The enhanced entry was due to increased virus deposition at the cell surface and not due to pelleting of the virus. Among the gD receptors, spinoculated GAG- HVEM cells showed restoration of HSV-1 entry compared to unspinoculated GAG+ HVEM cells. In contrast, spinoculated GAG- nectin-1 cells showed less entry than unspinoculated GAG+ nectin-1 cells. GAG- 3-O-sulfotransferase-expressing cells or heparinase treated GAG+ 3-O-sulfated heparan sulfate cells, in contrast, remained resistant to entry even after spinoculation. To investigate further, any potential effects of centrifugation on membrane fusion, a virus-free cell fusion assay was performed. Clearly, spinning had no effects on cell fusion, nor could it replace the need for all four essential glycoproteins. Taken together these results suggest that heparan sulfate plays a role of an attachment receptor, which could be substituted by spinoculation. This effect, however, varies with the gD receptor used, which in turn, could be used as a means for identifying gD receptor usage for entry into a cell type.
authors.affiliation: Department of Microbiology-Immunology, College of Medicine, University of Illinois at Chicago, Lions of Illinois Eye Research Institute, M/C 648, 1855 West Taylor Street, Chicago, IL 60612, USA.
authors.forename: Perry M
authors.initials: PM
authors.lastname: Scanlan
authors.forename: Vaibhav
authors.initials: V
authors.lastname: Tiwari
authors.forename: Susmita
authors.initials: S
authors.lastname: Bommireddy
authors.forename: Deepak
authors.initials: D
authors.lastname: Shukla
mesh_terms: D000818:Animals; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002498:Centrifugation / Q000379:methods; D006224:Cricetinae; D006025:Glycosaminoglycans / Q000172:deficiency / Q000378:metabolism; D006497:Heparan Sulfate / Q000172:deficiency* / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000378:metabolism / Q000472:pathogenicity*; D008561:Membrane Fusion; D000075983:Nectins; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D053240:Receptors, Tumor Necrosis Factor, Member 14; D011991:Receptors, Virus / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D014773:Virology / Q000379:methods
chemical_list: D015815:Cell Adhesion Molecules; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D000075983:Nectins; D018124:Receptors, Tumor Necrosis Factor; D053240:Receptors, Tumor Necrosis Factor, Member 14; D011991:Receptors, Virus; D014759:Viral Envelope Proteins; C000619010:NECTIN1 protein, human; C506028:TNFRSF14 protein, human; C049545:simplexvirus receptor