HVEM and nectin-1 are the major mediators of herpes simplex virus 1 (HSV-1) entry into human conjunctival epithelium.
OBJECTIVE
The human conjunctiva is a natural target for herpes simplex virus (HSV)-1 infection. The goals of this study were to investigate the cellular and molecular mechanisms of HSV-1 entry into human conjunctival epithelial (HCjE) cells. Specific features of entry studied included the method of initial viral binding to cells, pH dependency, and expression and usage of specific HSV-1 entry receptors.


METHODS
To observe HSV-1 initial binding, live cell imaging was performed on HSV-1-infected HCjE cells. Reporter HSV-1 virions expressing beta-galactosidase were used to determine entry of wild-type HSV-1(KOS) and a mutant, HSV-1(KOS)Rid1, into HCjE cells. HSV-1 replication in HCjE cells was determined by plaque assays. Lysosomotropic agents were used to determine whether viral entry was pH dependent. Reverse transcription (RT)-PCR, flow cytometry, and immunohistochemistry were used to determine the expression of receptors. Receptor-specific siRNAs were used to define the role of individual entry receptors.


RESULTS
HSV-1 virions attach to filopodia present on HCjE cells and use them to reach the cell body for entry. Cultured HCjE cells demonstrate susceptibility to HSV-1 entry and form plaques confirming viral replication. Blocking vesicular acidification significantly reduces entry, implicating a pH-dependent mode of entry. Multiple assays confirm the expression of entry receptors nectin-1, HVEM, and 3-O-sulfated heparan sulfate (3-OS HS) on the HCjE cell membrane. Knocking down of gD receptors by siRNAs interference implicates nectin-1 and HVEM as the major mediators of entry.


CONCLUSIONS
HSV-1 entry into HCjE cells is a pH-dependent process that is aided by targeted virus travel on filopodia. HCjE cells express all three major entry receptors, with nectin-1 and HVEM playing the predominant role in mediating entry.
Akhtar
Jihan
J
Departments of Ophthalmology and Visual Sciences, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA.
Tiwari
Vaibhav
V
Oh
Myung-Jin
MJ
Kovacs
Maria
M
Jani
Aarti
A
Kovacs
S Krisztian
SK
Valyi-Nagy
Tibor
T
Shukla
Deepak
D
D000818:Animals; D016466:CHO Cells; D003228:Conjunctiva / Q000502:physiology / Q000821:virology*; D006224:Cricetinae; D003412:Cricetulus; D004847:Epithelial Cells / Q000502:physiology / Q000821:virology*; D006561:Herpes Simplex / Q000517:prevention & control*; D018259:Herpesvirus 1, Human / Q000472:pathogenicity* / Q000502:physiology; D006801:Humans; D007381:Intermediate Filament Proteins / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000502:physiology*; D064231:Nestin; D011554:Pseudopodia / Q000821:virology; D053240:Receptors, Tumor Necrosis Factor, Member 14 / Q000502:physiology*; D010948:Viral Plaque Assay; D053586:Virus Internalization
D007381:Intermediate Filament Proteins; C577484:NES protein, human; D009419:Nerve Tissue Proteins; D064231:Nestin; D053240:Receptors, Tumor Necrosis Factor, Member 14; C506028:TNFRSF14 protein, human