title: Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry.
abstract: Human papillomaviruses (HPVs) infect epithelial cells and are associated with genital carcinoma. Most epithelial cell lines express cell-surface glycosaminoglycans (GAGs) usually found attached to the protein core of proteoglycans. Our aim was to study how GAGs influenced HPV entry. Using a human keratinocyte cell line (HaCaT), preincubation of HPV virus-like particles (VLPs) with GAGs showed a dose-dependent inhibition of binding. The IC(50) (50% inhibition) was only 0.5 microg/ml for heparin, 1 microg/ml for dextran sulfate, and 5-10 microg/ml for heparan sulfate from mucosal origin. Mutated chinese hamster ovary (CHO) cell lines lacking heparan sulfate or all GAGs were unable to bind HPV VLPs. Here we also report a method to study internalization by using VLPs labeled with carboxy-fluorescein diacetate, succinimidyl ester, a fluorochrome that is only activated after cell entry. Pretreatment of labeled HPV VLPs with heparin inhibited uptake, suggesting a primary interaction between HPV and cell-surface heparan sulfate.
authors.affiliation: Department of Virology, Umeå University, SE-901 85 Umeå, Sweden.
authors.forename: Peter
authors.initials: P
authors.lastname: Drobni
authors.forename: Nitesh
authors.initials: N
authors.lastname: Mistry
authors.forename: Nigel
authors.initials: N
authors.lastname: McMillan
authors.forename: Magnus
authors.initials: M
authors.lastname: Evander
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D005452:Fluoresceins; D005453:Fluorescence; D006025:Glycosaminoglycans / Q000502:physiology; D006493:Heparin / Q000494:pharmacology; D019763:Heparin Lyase / Q000494:pharmacology; D006497:Heparan Sulfate / Q000502:physiology*; D006801:Humans; D015603:Keratinocytes / Q000821:virology; D027383:Papillomaviridae / Q000502:physiology*; D014771:Virion / Q000502:physiology*
chemical_list: D005452:Fluoresceins; D006025:Glycosaminoglycans; D006493:Heparin; D019763:Heparin Lyase; D006497:Heparan Sulfate; C018506:diacetylfluorescein