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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY021558-09
Application #
9933057
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mckie, George Ann
Project Start
2011-09-30
Project End
2021-06-30
Budget Start
2020-07-01
Budget End
2021-06-30
Support Year
9
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Massachusetts Eye and Ear Infirmary
Department
Type
DUNS #
073825945
City
Boston
State
MA
Country
United States
Zip Code
02114
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Uchino, Yuichi; Woodward, Ashley M; Mauris, Jérôme et al. (2018) Galectin-3 is an amplifier of the interleukin-1?-mediated inflammatory response in corneal keratinocytes. Immunology 154:490-499
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Lee, Jeong Yoon; Lee, Ji Sun; Materne, Emma C et al. (2018) Bacterial RecA Protein Promotes Adenoviral Recombination during In Vitro Infection. mSphere 3:
Ismail, Ashrafali M; Lee, Ji Sun; Lee, Jeong Yoon et al. (2018) Corrigendum: Adenoviromics: Mining the Human Adenovirus Species D Genome. Front Microbiol 9:3005
Ismail, Ashrafali M; Lee, Ji Sun; Lee, Jeong Yoon et al. (2018) Adenoviromics: Mining the Human Adenovirus Species D Genome. Front Microbiol 9:2178
Pan, Haibin; Yan, Yuqian; Zhang, Jing et al. (2018) Rapid Construction of a Replication-Competent Infectious Clone of Human Adenovirus Type 14 by Gibson Assembly. Viruses 10:
Ismail, Ashrafali M; Cui, Tiange; Dommaraju, Kalpana et al. (2018) Genomic analysis of a large set of currently-and historically-important human adenovirus pathogens. Emerg Microbes Infect 7:10
Sié, Ali; Diarra, Abdramane; Millogo, Ourohiré et al. (2018) Seasonal and Temporal Trends in Childhood Conjunctivitis in Burkina Faso. Am J Trop Med Hyg 99:229-232

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