Caveolin-1 Associated Adenovirus Entry into Human Corneal Cells

Abstract

The cellular entry of viruses represents a critical area of study, not only for viral tropism, but also because viral entry dictates the nature of the immune response elicited upon infection. Epidemic keratoconjunctivitis (EKC), caused by viruses within human adenovirus species D (HAdV-D), is a severe, ocular surface infection associated with corneal inflammation. Clathrin-mediated endocytosis has previously been shown to play a critical role in entry of other HAdV species into many host cell types. However, HAdV-D endocytosis into corneal cells has not been extensively studied. Herein, we show an essential role for cholesterol rich, lipid raft microdomains and caveolin-1, in the entry of HAdV-D37 into primary human corneal fibroblasts. Cholesterol depletion using methyl-β-cyclodextrin (MβCD) profoundly reduced viral infection. When replenished with soluble cholesterol, the effect of MβCD was reversed, allowing productive viral infection. HAdV-D37 DNA was identified in caveolin-1 rich endosomal fractions after infection. Src kinase activity was also increased in caveolin-1 rich endosomal fractions after infection, and Src phosphorylation and CXCL1 induction were both decreased in caveolin-1-/- mice corneas compared to wild type mice. siRNA knock down of caveolin-1 in corneal cells reduced chemokine induction upon viral infection, and caveolin-1-/- mouse corneas showed reduced cellular entry of HAdV-D37. As a control, HAdV-C2, a non-corneal pathogen, appeared to utilize the caveolar pathway for entry into A549 cells, but failed to infect corneal cells entirely, indicating virus and cell specific tropism. Immuno-electron microscopy confirmed the presence of caveolin-1 in HAdV-D37-containing vesicles during the earliest stages of viral entry. Collectively, these experiments indicate for the first time that HAdV-D37 uses a lipid raft mediated caveolin-1 associated pathway for entry into corneal cells, and connects the processes of viral entry with downstream proinflammatory cell signaling.

Recombination of the epsilon determinant and corneal tropism: Human adenovirus species D types 15, 29, 56, and 69

“…Because human adenoviruses do not replicate in mouse cells (Blair et al, 1989; Ginsberg et al, 1991; Silverstein and Strohl, 1986; Zucker and Flint, 1985), the mouse adenovirus keratitis model is an inflammatory rather than a replication model and bypasses the adenoviral replication-resistant mouse corneal epithelium. We have shown previously that tropism in humans is better predicted by assays of HAdV entry into human corneal epithelial cells, than by inflammatory responses upon injection of virus into the mouse corneal stroma (Yousuf et al, 2013; Zhou et al, 2012). Therefore, we tested the cellular entry of Cy3 labeled virus in Tert-immortalized human corneal epithelial (THE) cells (gift of Dr. Jerry Shay) and primary human corneal fibroblasts (HCF), with A549 cells as controls.…”

Caveolins and caveolae in ocular physiology and pathophysiology

“…However, it remains an open question if caveolae transcytosis plays a role in pathological BRB breakdown that deserves careful study. Finally, caveolae seem to play important roles at the ocular surface both in pathogen uptake (Mukherjee et al, 2015; Yousuf et al, 2013), corneal inflammation (Reidy et al, 2013), and wound healing (Amino et al, 1997; Rhim et al, 2010) but these areas are yet to receive further attention.…”

“…Recent work indicates that adenoviruses utilize a caveolae-mediated pathway for entry into corneal cells (Mukherjee et al, 2015; Yousuf et al, 2013). Importantly, disruption of caveolae and cholesterol-rich membrane domains by cholesterol depletion dramatically reduced viral infection (Yousuf et al, 2013).…”

“…Recent work indicates that adenoviruses utilize a caveolae-mediated pathway for entry into corneal cells (Mukherjee et al, 2015; Yousuf et al, 2013). Importantly, disruption of caveolae and cholesterol-rich membrane domains by cholesterol depletion dramatically reduced viral infection (Yousuf et al, 2013). Studies designed to examine viral-mediated gene transfer to the corneal epithelium revealed improved transduction efficiency of recombinant adenovirus to the corneal epithelium by the PDE5 inhibitor, sildenafil, which acted by redistributing caveolin-1 expression from the normal basal epithelial location to suprabasal epithelium thus facilitating viral uptake (Saghizadeh et al, 2010).…”

Novel model of innate immunity in corneal infection

“…We also previously reported that Src kinase is upregulated in the mouse adenovirus keratitis model (Yousuf et al 2013) and may play a role in CXC chemokine expression. Using chemical signaling inhibitors to study signaling and innate immunity in an animal model is a challenge, and sometimes not feasible.…”