Extracellular Matrix Determinants of Adenovirus Replication in the Human Cornea

Abstract

Abstract: : Purpose: Adenoviruses (Ad) typically demonstrate specific tissue tropisms, for example, the association of Ad19 with adenovirus keratitis. Because internalization of adenoviruses by host cells is partly dependent upon specific cell surface integrins that normally bind extracellular matrix constituents, we sought to determine if corneal extracellular matrix components influence the apparent tissue tropism of Ad19 for the human cornea. Methods: Organotypically cultured human corneas were infected in vitro with Ad19 and the non-corneatropic Ad2, and viral growth titers compared after 7 days of infection. Various Ad serotypes were compared for their capacity to replicate in human corneal epithelial cells (HCEC) in monolayer culture. Viral growth of Ad19 and Ad2 were then compared in HCEC cultured on different extracellular matrix substrates. Results: In organotypic corneal organ cultures, Ad2 titers rose minimally or fell below the sensitivity of the assay, but Ad19 titers increased 30-fold. Growth of HCEC on vitronectin, but not other substrates, reduced Ad2 replication compared to plastic and poly-D-lysine controls (respective mean TCID/cell: 10² vs. 10⁴, 10⁴), and enhanced Ad19 replication (10⁵ vs. 10³, 10³). Increased α_v integrin as well as relatively greater tyrosine kinase phosphorylation were demonstrated by Western blot analysis of HCEC cultured on vitronectin as compared to plastic. Conclusions: Given the requirement of adenoviruses for specific cell surface integrins in order to enter host cells, we hypothesize that differential tissue tropisms for adenoviruses may derive in part from tissue-specific extracellular matrix expression. In vitro, vitronectin enhances growth of Ad19, possibly by upregulation of α_v, part of the vitronectin receptor integrin pairs, α_vß₃ and α_vß₅. Adenovirus internalization is also protein kinase-dependent; increased phosphorylation of tyrosine kinases in HCEC, possibly due to binding and aggregation of cell surface integrins by vitronectin, likely enhances Ad19 entry and subsequent replication.

Keywords: adenovirus • cornea: epithelium • extracellular matrix