Corneal disease is one of the major causes of blindness. Corneal infections include epidemic keratoconjunctivitis (EKC), caused by human adenoviruses (HAdV). EKC is characterized by a severe, acute keratoconjunctivitis. HAdV species D, in particular, types HAdV-D8, 37, 64 (formerly type 19), and the recently identified 53, 54 and 56, cause EKC, the only adenoviral infection with significant corneal involvement. Detailed knowledge of the foremost events in infection, viral entry and intracellular trafficking, are needed to design a specific therapy for the disorder. Our prior work focused on human corneal fibroblasts, because of their role in corneal inflammation. We now extend our studies to corneal epithelial cells, the first cells in the cornea to encounter virus.
The Specific Aims of this proposal are to test the hypotheses that 1) adenovirus entry and trafficking in corneal epithelial cells requires clathrin mediated endocytosis, 2) adenovirus infection of corneal epithelial cells is controlled by dynamin 2, and 3) signaling molecules unique to clathrin vesicles and caveosomes determine adenovirus trafficking in corneal epithelial cells and fibroblasts, respectively. Viral entry is a complex phenomenon, and while governed by the tropism of the infecting virus, is also cell type dependent. Our preliminary data in human corneal epithelial cells suggests a critical role for clathrin mediated endocytosis in HAdV-D37 infection. We will apply chemical inhibitors and siRNA against specific components of each viral entry pathway to dissect and elucidate the mechanisms of HAdV-D37 entry and trafficking in primary human corneal epithelial cells. Dynamin 2 shows a paradoxically negative effect on HAdV-D37 entry in corneal fibroblasts, and preliminary studies suggest a similar effect in corneal epithelial cells. We will test viral entry when overexpressing various dynamin 2 mutants in order to determine the mechanism by which dynamin 2 controls infection. Finally, because endosomes are a platform for receptor mediated signaling distinct from membrane signaling and play an essential role in antiviral responses, we will purify endosomes from infected and non-infected corneal cells and perform mass spectrometry analysis to elucidate the specific signalosomes activated by viral infection. The role of validated proteins in viral trafficking and replication will be tested using inhibitors and siRNA techniques. The proposed studies will take us closer to our goal of effective information-based therapies against EKC, a common affliction. Therefore, the proposed study addresses a major public health concern.
Adenovirus is the most common cause of human eye infection. This project will study human corneal epithelial cells to elucidate the means of entry and intracellular trafficking for human adenovirus type 37, associated with epidemic ocular surface infection and the most virulent adenoviral eye pathogen.
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