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Jaya Rajaiya, JiSun Lee, AshrafAli M Ismail, JeongYoon Lee, James Chodosh; Differential trafficking of adenovirus in corneal epithelial cells and fibroblasts. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2335.
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© ARVO (1962-2015); The Authors (2016-present)
Ocular surface infection by viruses within human adenovirus species D (HAdV-D) causes epidemic keratoconjunctivitis, manifest by acute membranous keratoconjunctivitis and delayed-onset stromal keratitis. We have earlier shown that human adenovirus type D37 (HAdV-D37) uses a lipid raft mediated caveolin-1 pathway to enter human corneal fibroblasts (HCF), and that this pathway is negatively regulated by dynamin-2. In the current study, we extend these studies to the role of cellular microtubules in viral entry, and further determine mechanisms of HAdV-D37 entry into tert-immortalized human corneal epithelial (THE) cells.
HCF and THE cells were grown in standard media, and pretreated with bafilomycin, cytochalasin, nocodazole, IPA-3, or DMSO control for 30 min and then infected with HAdV-D37 for 1 hr. In other experiments, cells were transfected with siRNA against dynamin-2, or scRNA, prior to infection. RNA was extracted by Trizol for real-time qPCR or cells lysed for protein studies. Viral titers were performed by the tissue culture infectious dose assay. Alternately, cell cultures were fixed with paraformaldehyde for confocal microscopy to visualize viral entry and changes in intracellular microtubules.
The endosomal acidification inhibitor, bafilomycin did not impact viral entry in either cell type. The actin polymerization inhibitor, cytochalasin D, inhibited viral entry in both cell types. The microtubule inhibitor, nocodazole, repressed viral entry in HCF, but only partially inhibited viral entry in THE cells. The macropinocytosis inhibitor, IPA-3, significantly decreased adenoviral infection in THE cells. Pretreatment with dynamin-2 siRNA increased HAdV-D37 entry and replication, increased Src phosphorylation, and caused a concurrent change in microtubule organizing center localization in HCF, as shown by confocal immunomicroscopy with antibody to pericentrin, but had no effect in THE cells.
Adenovirus trafficking differs among cell types within the cornea. Understanding viral entry pathways in adenovirus keratitis may lead to new, information-based molecular therapies against infection.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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