Abstract
Purpose: :
Epidemic keratoconjunctivitis (EKC), caused by human adenovirus (HAdV) types 8, 19, 37, and 53, is a severe, hyperacute, ocular surface infection associated with prolonged stromal keratitis. Signaling molecules play a pivotal role in HAdV pathogenesis, from pathogen entry to host cell defense. Our earlier studies suggested that HAdV infection of stromal keratocytes activates key signaling molecules, Src, FAK, and PI3K, that in turn regulate chemokine expression in the corneal stroma. Signalosomes sequestered in cholesterol-rich microdomains, lipid rafts (LR), while important for the entry of some viruses, have not been extensively studied in adenoviral infection.
Methods: :
Prior to infection, LR in keratocytes were disrupted by methyl β-cyclodextrin (MβCD; 5 mM for 1 hr). Control cultures were treated with arginine-glycine-aspartic acid (RGD) monomers to block viral internalization, or PP2 (chemical inhibitor of Src kinases), previously shown to inhibit chemokine expression by infected cells. Keratocytes were then infected with Cy3 labeled HAdV-19 for 30 min and 1 hr and analyzed by confocal microscopy. Real-time PCR was performed to determine viral gene expression as an indirect confirmation of viral entry. After infection, LR fractions were isolated by detergent free methods using a sucrose gradient, and immunoblotted to identify phosphorylated Src in LR and non-LR fractions.
Results: :
In untreated cells, the presence of virus in the cytoplasm was evident as early as 30 min post infection. In MβCD treated cells, virus remained localized to the cell membrane even at 1 hr post infection, suggesting impairment of viral entry. Treatment with RGD or PP2 also reduced viral internalization as compared to controls. Phosphorylated Src was increased in LR after viral infection as compared to mock or MβCD treated cells. Interestingly, addition of RGD peptide monomer also reduced phosphorylation of Src and diminished viral entry and subsequent viral gene expression.
Conclusions: :
These results strongly suggest the presence and activity of phosphorylated Src in the LR of adenovirus infected cells, and confirm a role for LR in adenovirus ocular pathogenesis.
Keywords: adenovirus • cornea: stroma and keratocytes • signal transduction