Novel Mechanisms in Adenoviral Ocular Pathogenesis

PROJECT SUMMARY Conjunctivitis impacts approximately 6 million people annually in the United States, accounting for 1% of all primary care office visits, and costing 430 million USD annually. Viruses are responsible for ~80% of conjunctivitis cases, and human adenoviruses (HAdVs) are implicated in up to 65% of these. A particularly severe cause of conjunctivitis, epidemic keratoconjunctivitis (EKC) is caused principally by viruses within human adenovirus species D (HAdV-D), for example the highly virulent adenovirus type 37 (HAdV-D37). The clinical manifestations of EKC include severe membranous conjunctivitis and epithelial keratitis, followed by delayed onset of multifocal subepithelial (stromal) corneal infiltrates. The latter cause light sensitivity and reduced vision, and can persist or recur for months to years after the acute infection, long after the virus is gone. We have shown in work published over the last 20+ years that adenovirus binding to human corneal cells activates complex intracellular signaling events that control subsequent cellular entry, intracellular trafficking of virions for delivery of viral DNA into cell nuclei, virion assembly and viral replication, and proinflammatory gene expression by infected cells. In this application, we extend our studies with new revelations on viral trafficking, new functions for a viral structural protein, and new evidence for expression of a novel proinflammatory mediator by infected corneal epithelial cells. The specific aims of this proposal are to test the hypotheses that 1) kinesin-1 and V-ATPase play previously unidentified roles in adenovirus trafficking, 2) adenoviral minor coat protein IIIa promotes adenovirus replication, and 3) corneal epithelial cell HMGB1 promotes inflammation in adenovirus keratitis. We will apply proteomic, siRNA, and imaging approaches to determine final stages of virion uncoating in human corneal epithelial cells and human corneal fibroblasts. A role for viral protein IIIa in nuclear transport of viral proteins for virion assembly will be determined by use of deletion and point mutants of pIIIa through surface plasmon resonance, imaging, PCR, and proteomics experiments. Finally, we will determine a role for human corneal epithelial cell expression of the multi-potent molecule HMGB1 in the formation of subepithelial infiltrates in EKC. These studies are designed to move us toward a more detailed understanding of adenoviral corneal pathogenesis, in order to reach our goal of an information-based therapy to mitigate the morbidity of adenovirus ocular infections.