title: HSV-1 infection of human corneal epithelial cells: receptor-mediated entry and trends of re-infection.
abstract: OBJECTIVE
  The human cornea is a primary target for herpes simplex virus-1 (HSV-1) infection. The goals of the study were to determine the cellular modalities of HSV-1 entry into human corneal epithelial (HCE) cells. Specific features of the study included identifying major entry receptors, assessing pH dependency, and determining trends of re-infection.
  
  
  METHODS
  A recombinant HSV-1 virus expressing beta-galactosidase was used to ascertain HSV-1 entry into HCE cells. Viral replication within cells was confirmed using a time point plaque assay. Lysosomotropic agents were used to test for pH dependency of entry. Flow cytometry and immunocytochemistry were used to determine expression of three cellular receptors--nectin-1, herpesvirus entry mediator (HVEM), and paired immunoglobulin-like 2 receptor alpha (PILR-a). The necessity of these receptors for viral entry was tested using antibody-blocking. Finally, trends of re-infection were investigated using viral entry assay and flow cytometry post-primary infection.
  
  
  RESULTS
  Cultured HCE cells showed high susceptibility to HSV-1 entry and replication. Entry was demonstrated to be pH dependent as blocking vesicular acidification decreased entry. Entry receptors expressed on the cell membrane include nectin-1, HVEM, and PILR-α. Receptor-specific antibodies blocked entry receptors, reduced viral entry and indicated nectin-1 as the primary receptor used for entry. Cells re-infected with HSV-1 showed a decrease in entry, which was correlated to decreased levels of nectin-1 as demonstrated by flow cytometry.
  
  
  CONCLUSIONS
  HSV-1 is capable of developing an infection in HCE cells using a pH dependent entry process that involves primarily nectin-1 but also the HVEM and PILR-α receptors. Re-infected cells show decreased levels of entry, correlated with a decreased level of nectin-1 receptor expression.
authors.affiliation: Department of Ophthalmology and Visual Sciences, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA.
authors.forename: Arpeet
authors.initials: A
authors.lastname: Shah
authors.forename: Asim V
authors.initials: AV
authors.lastname: Farooq
authors.forename: Vaibhav
authors.initials: V
authors.lastname: Tiwari
authors.forename: Min-Jung
authors.initials: MJ
authors.lastname: Kim
authors.forename: Deepak
authors.initials: D
authors.lastname: Shukla
mesh_terms: D019138:Antibodies, Blocking / Q000276:immunology; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D015536:Down-Regulation / Q000235:genetics; D004847:Epithelial Cells / Q000821:virology*; D019573:Epithelium, Corneal / Q000166:cytology*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D018259:Herpesvirus 1, Human / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016849:Keratitis, Herpetic / Q000473:pathology / Q000821:virology*; D000075983:Nectins; D011991:Receptors, Virus / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053586:Virus Internalization; D014779:Virus Replication / Q000502:physiology
chemical_list: D019138:Antibodies, Blocking; D015815:Cell Adhesion Molecules; C000619010:NECTIN1 protein, human; D000075983:Nectins; D011991:Receptors, Virus