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J. Akhtar, M. Kovacs, M.-J. Oh, V. Tiwari, A. Jani, D. Shukla; Novel Aspects of Herpes Simplex Virus 1 Entry Into Human Conjunctival Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5517. doi: https://doi.org/.
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The goal of this study was to study unique aspects of herpes simplex virus-1 (HSV-1) entry into human conjunctival epithelial (HCjE) cells. Specific features of entry that were studied include method of initial viral binding, usage of specific HSV-1 entry receptors, and pH dependency of entry.
To observe HSV-1 initial binding, live cell imaging was utilized. Viral entry assays determined entry of β-galactosidase-expressing recombinant HSV-1(KOS)gL86 and HSV-1(KOS)Rid1tk12, a mutant of HSV-1, into HCjE cells. HSV-1 replication was determined by plaque assays. RT-PCR and flow cytometry were used for identifying the specific receptors responsible for HSV-1 entry into HCjE cells. The receptor expression data was confirmed by confocal microscopy. Receptor specific siRNAs were used to probe the role of individual receptors in the entry process. Lysosomotropic agents such as bafilomycin A1 (BFLA-1) and chloroquine were use to determine the pH dependency of viral entry into HCjE cells.
HSV-1 binds to HCjE filopodia and uses filopodia to "surf" towards cell membrane before infection. HCjE cells are susceptible to HSV-1 entry and show plaque formation, confirming viral replication. RT-PCR, flow cytometry, and confocal microscopy confirm the expression of entry receptors nectin-1, HVEM, and 3-O-sulfated heparan sulfate (3-OS HS) on HCjE cell membrane and siRNA interference of receptor production decreases viral entry. Blocking vesicular acidification during viral entry also significantly reduced viral entry.
HSV-1 entry into HCjE cells is a pH dependent process that requires filopodial surfing. HCjE cells express and likely utilize all three major entry receptors for mediating HSV-1 entry. Identification of the receptors and the mode of entry will aid to future therapies to treat infection of the conjunctiva.
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