HSV-1 infection of human corneal epithelial cells: receptor-mediated entry and trends of re-infection

Abstract

Purpose: 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.

Figure 1

SV-1 can enter into culture HCE cells. A: Dose response curve of HSV-1 entry into HCE cells. Cultured HCE cells along with naturally HSV-1 resistant CHO-K1 cells were plated in 96-well culture dishes and inoculated with twofold serial dilutions of β-galactosidase-expressing recombinant HSV-1(KOS)gL86 virus at the plaque forming units (PFU) indicated. After 6 h, the cells were washed, permeablized and incubated with ONPG substrate. Viral entry was measured using a spectrophotometer which measured beta-galactosidase activity at an optical density of 410 nm. Values in the figure were plotted as the mean of three determinations (±SD). B: Confirmation of HSV-1 entry into HCE cells by X-gal staining. HCE cells grown (4×10⁶ cells) in six well dishes were inoculated with β-galactosidase-expressing HSV-1(KOS)gL86 virus at 20 PFU/cell. CHO-K1 cells were also infected in parallel as a negative control. After 6 h of infection at 37 °C, cells were washed, fixed, and permeabilized. X-gal was then added which yields an insoluble blue product upon hydrolysis by β-galactosidase. Blue cells, which represent cells with viral entry, were seen in HCE cells, but not the naturally resistant CHO-K1 cells. Microscopy was performed using a 20X objective of the Zeiss Axiovert 100 microscope. The slide book version 3.0 was used for the images.

Figure 2

HSV-1 can successfully grow and replicate in HCE cells. Confluent monolayers of HCE, Vero, and naturally-resistant CHO-K1 cells were infected with HSV-1(KOS)804 at an MOI of 0.01 for 90 min at 37 °C. Innoculums were harvested, fixed, and stained at 0, 12, 18, 24, and 36 h. Data represents the mean of three samples (±SD). A typical plaque formed by Vero and HCE cells stained with crystal violet at 18 h of infection with HSV-1(KOS)804 virus is shown in (B) and (C) accordingly. Plaques were not formed in the HSV-1 resistant CHO-K1 and the lack of plaques are shown in D. Increasing plaque size over time demonstrating progressive infection was confirmed in E.

Figure 3

HSV-1 entry into HCE cells is a pH dependent process. Monolayers of HCE cells were either mock treated (no treatment) or treated with indicated concentrations of bafilomycin (A) or chloroquine (B) and exposed to HSV-1(KOS)gL86. Viral entry was measured using a spectrophotometer at an OD of 410 nm and plotted as the mean of three samples (±SD).

Figure 4

Quantitative RT–PCR analysis for entry receptor expression. Total RNA was isolated from HCE and CHO-K1 cells and converted to cDNA and analyzed by PCR for the receptors as indicated. A house keeping gene, β-actin (ACTB), was used as a control (A). Densitometry analysis was performed using the NIH ImageJ software (Version 1.43) and data for relative intensity was plotted as a histogram (B).

Figure 5

Immunofluorescence imaging of receptors on HCE cell membrane. Images shown were taken using the FITC filter of confocal microscope (Leica SP20). Cells were blocked for 90 min, washed, and then either mock treated with buffer alone (B, D, F) or treated with primary antibodies for Nectin-1 (A), HVEM (C), and PILR-alpha (E). Images were taken after the incubation of HCE cells with FITC-conjugated secondary antibodies. Staining of cells with green demonstrate receptor expression.

Figure 6

Flow cytometry analysis of cell-surface receptor expression. Expression was detected by Fluorescence-activated cell sorter (FACS) analysis. Cells were treated with primary antibodies to Nectin-1 (A), HVEM (B), or PILR-alpha (C). HCE cells stained only with FITC-conjugated secondary antibody were used as background controls and are shown as the dark gray in the figure.

Figure 7

Antibodies to the major receptors block HSV-1 entry. Monolayers of cells plated in 96-well culture dishes were incubated with serial dilutions of primary antibodies to Nectin-1, HVEM, and PILR-α for 2 h. Cells were then exposed to HSV-1(KOS)gL86 virus and viral entry was measured 6 h post-infection using a spectrophotometer at an OD of 410 nm. The ratio of entry between cells treated and those untreated are reported as percent blocking. Combinations of antibodies that block these receptors were also studied. Data shown are the means of triplicate determinations (±SD). Anti-nectin-1 (A), anti-HVEM (B), anti-PILR-α (C), a combination of anti-nectin-1 and anti-HVEM antibodies (D) and a combination of all three antibodies (E).

Figure 8

Superinfected cells demonstrate less viral entry. Monolayers of cells plated in 96-well culture dishes were treated with a primary viral dose of HSV-1(KOS) at an MOI of 0.01. After 48 h of incubation at 37 °C, cell were treated with serial dilution of a secondary viral dose of HSV-1(KOS)gL86. Viral entry was measured using a spectrophotometer at an OD of 410 nm. Cells solely infected with only one dose of HSV-1(KOS)gL86 were used as control.

Figure 9

Cells demonstrate a decreased level of Nectin-1 receptor expression after primary infection. Monolayers of HCE cells plated on a 6-well tissue culture dish were treated with a viral dose at an MOI of 0.01 and incubated for 48 h at 37 °C. Cells were then treated with MTT to distinguish live from dead cells. FACS was then performed to test levels of Nectin-1 on living infected cells tagged with red fluorescent protein. HCE cells untreated with a viral dose were used as a control and are shown as the green shade.