Entry of Epidemic Keratoconjunctivitis-Associated Human Adenovirus Type 37 in Human Corneal Epithelial Cells

Abstract

Purpose: Ocular infection by human adenovirus species D type 37 (HAdV-D37) causes epidemic keratoconjunctivitis, a severe, hyperacute condition. The corneal component of epidemic keratoconjunctivitis begins upon infection of corneal epithelium, and the mechanism of viral entry dictates subsequent proinflammatory gene expression. Therefore, it is important to understand the specific pathways of adenoviral entry in these cells.

Methods: Transmission electron microscopy of primary and tert-immortalized human corneal epithelial cells infected with HAdV-D37 was performed to identify the means of viral entry. Confocal microscopy was used to determine intracellular trafficking. The results of targeted small interfering RNA and specific chemical inhibitors were analyzed by quantitative PCR, and Western blot.

Results: By transmission electron microscopy, HAdV-D37 was seen to enter by both clathrin-coated pits and macropinocytosis; however, entry was both pH and dynamin 2 independent. Small interfering RNA against clathrin, AP2A1, and lysosome-associated membrane protein 1, but not early endosome antigen 1, decreased early viral gene expression. Ethyl-isopropyl amiloride, which blocks micropinocytosis, did not affect HAdV-D37 entry, but IPA, an inhibitor of p21-activated kinase, and important to actin polymerization, decreased viral entry in a dose-dependent manner.

Conclusions: HAdV-D37 enters human corneal epithelial cells by a noncanonical clathrin-mediated pathway involving lysosome-associated membrane protein 1 and PAK1, independent of pH, dynamin, and early endosome antigen 1. We showed earlier that HAdV-D37 enters human keratocytes through caveolae. Therefore, epidemic keratoconjunctivitis-associated viruses enter different corneal cell types via disparate pathways, which could account for a relative paucity of proinflammatory gene expression upon infection of corneal epithelial cells compared with keratocytes, as seen in prior studies.

Figure 1.

Transmission electron microscopy of HAdV-D37-infected PHCE (A–G) and THE cells (H–M). (A) PHCE expression of cytokeratin marker 3/12 was performed to confirm a corneal epithelial phenotype. DAPI staining was included to show cell nuclei. In representative images from 15 minutes post infection, PHCE cells show a virion (arrow) (B and H) within a clathrin pit like structure, and viruses within a membrane ruffle (E and K), suggesting uptake by macropinocytosis. By 30 minutes hpi (C, F, I, and L), virions can be seen in individual intracellular endosomes in both PHCE and THE cells, and by 1 hpi (D, G, J, and M) within vesicles containing multivesicular bodies (MVB). Scale bar = 200 microns.

Figure 2.

HAdV-D37 enters THE cells via a clathrin mediated pathway. (A) Confocal microscopy of THE cells pretreated with scRNA (top) or siClathrin (bottom) show a reduction in Cy3 labelled HAdV-D37 (red) in siClathrin treated cells at 2 hpi. Phalloidin staining is shown in green. (B) Clathrin knockdown, as shown by Western blotting and mRNA expression via RT-qPCR quantification. Reduced E1A gene expression is evident by RT-qPCR in clathrin knock down cells (^*P ≤ 0.05). (C) Confirmation of clathrin mediated adenoviral entry using transferrin control, panels 1, 2, and 3; insets show co-localization in the merged image between clathrin (green) and Cy3-HAdV-D37 (red), between transferrin (green) and Cy3-HAdV-D37 (red), and between clathrin (green) and transferrin (red) respectively, at 1 hpi.

Figure 3.

Dynamin-independent clathrin-mediated pathway. (A) Confocal images reveal no apparent difference in entry of Cy3-HAdV-D37 (red) between scRNA (top) and dynamin 2 siRNA (siDNM2) treated (bottom) THE cells at 2 hpi. Acetylated tubulin staining is shown in green. (B) Western blot showing knock down of dynamin 2, along with GAPDH loading control. Successful dynamin 2 knock down was confirmed by RT-qPCR. E1A gene expression at 2 hpi by RT-qPCR showed no difference between control and siDNM2 knock down cells.

Figure 4.

AP2A1-dependent, epsin 1–independent pathway. (A) Confocal images reveal successful entry of Cy3-HAdV-D37 (red) into scRNA treated cells. However, entry was blocked in siAP2A1 treated cells. Phalloidin staining is shown in green. (B) Western blot showing AP2A1 knock down, with GAPDH shown as loading control. Bar graphs show mRNA levels after AP2A1 knock down, and near complete abrogation of E1A gene expression in siAP2A1-treated cells (^*P ≤ 0.05). (C) Epsin1 knock down, as confirmed by RT-qPCR, did not reduce HAdV-D37 E1A gene expression.

Figure 5.

HAdV-D37 endosomal trafficking is not canonical. (A) EEA1 knock down shown by Western blot, and RT-qPCR (upper left bar graph). E1A gene expression was not reduced by siEEA1 pretreatment (lower left bar graph). (B) LAMP1 knock down shown by Western blot and RT-qPCR (upper right bar graph). E1A gene expression was reduced upon siLAMP1 treatment (lower right bar graph) (^*P ≤ 0.05).

Figure 6.

Actin is important for HAdV-D37 entry. (A) Both cytochalasin inhibitors D and B decreased E1A gene expression in a dose-dependent manner (^*P ≤ 0.05). (B) Bafilomycin A, an inhibitor of endosome acidification did not reduce E1A gene expression even at high concentration. (C) The macropinosome formation inhibitor, EIPA, had no effect on E1A gene expression. (D) The Pak1 inhibitor, IPA3, decreased E1A gene expression in a dose-dependent manner (^*P ≤ 0.05).

Figure 7.

Clathrin-mediated entry in PHCE cells. (A) Open bar shows clathrin mRNA expression; solid bar shows E1A mRNA expression. (B) Western blot of clathrin and GAPDH load control. (C) Open bar shows AP2A1 mRNA expression; solid bar shows E1A mRNA expression. (D) Western blot of AP2A1 and GAPDH load control. (E) Open bar shows dynamin 2 (DNM2) mRNA expression; solid bar shows E1A mRNA expression. (F) Western blot of dynamin 2 and GAPDH load control. ^*P = 0.01, Student t-test, comparing E1A mRNA expression between scRNA- and siRNA-treated cells.

Figure 8.

HAdV-D37 does not use caveolin to enter human corneal epithelial cells. (A) Confocal images in merge tile did not show co-localization of caveolin 1 (green) and Cy3-HAdV-D37 (red). (B) Caveolin 1 knock down as shown by Western blot and RT-qPCR (upper bar graph) did not decrease E1A gene expression (lower bar graph). Experiments were performed at 2 hours after infection.

Figure 9.

Schematic model of HAdV-D37 entry into human corneal epithelial cells. CCP, clathrin-coated pit; MVB, multivesicular body; LE, late endosome; M, macropinosome; MTs, microtubules; MTOC, microtubule organizing center.