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Human adenovirus type 26 infection of epithelial cells is αvβ3 integrin, clathrin and caveolin-1 mediated

PURPOSE OF WORK. Due to its low seroprevalence, species D human adenovirus type 26 (HAdV26), has been considered a promising vector for the development of vaccines. The basic biology of this virus with regard to receptor use, cell attachment, internalization, and intracellular trafficking is still poorly understood. In our work we investigated which molecules govern HAdV26 endocytosis and transduction efficiency in human epithelial cells. MATERIALS AND METHODS. The knockdown of protein of interest was done by transient transfection of siRNA. Cell clones with altered expression of molecules of interest were obtained by stable transfection. Flow cytometry and confocal microscopy were used to study adenovirus transduction efficiency and internalization. Western blot was done by using standard protocols. RESULTS. In this work, we investigated the roles of CAR, CD46, αv integrins, dynamin-2, clathrin and caveolin- 1 in HAdV26 infection of human epithelial cell lines. By performing different gain- and loss- of-function studies, we found that αvβ3 integrin is required for efficient infection of epithelial cells by HAdV26. By studying intracellular trafficking of fluorescently labelled HAdV26 in A549 cells and A549-derived cell clones with stably increased expression of αvβ3 integrin, we observed that HAdV26 colocalizes with αvβ3 integrin and that increased αvβ3 integrin enhances internalization of HAdV26. Downregulation of clathrin caused increased cell surface expression of αvβ3 integrin and therefore increased both internalization and transduction efficacy of HAdV26 in A549 cells. Inhibition of clathrin- mediated endocytosis by using specific inhibitor had the same effect, namely increased transduction efficiency of HAdV26. Downregulation of caveolin-1 also increased cell surface expression of αvβ3 integrin, however had no effect on HAdV26 transduction efficiency, but rather caused slight decrease in HAdV26 internalization. Contrary to adenovirus type 5 and 35, downregulation of dynamin-2 did not cause significant decrease in HAdV26 transduction efficiency. CONCLUSION. Based on our results we conclude that: i) HAdV26 uses αvβ3 integrin as a receptor for infecting epithelial cells, ii) clathrin knockdown increases amount of αvβ3 integrin on cell surface therefore allowing increased HAdV26 transduction efficiency, iii) both clathrin and caveolin-1 are needed for functional trafficking of HAdV26. Further studies aimed at detailed description of HAdV26 endocytosis are ongoing. MAIN BIBLIOGRAPHY. Nestić D, Uil TG, Ma J, Roy S, Vellinga J, Baker AH, Custers J, Majhen D. αvβ3 Integrin Is Required for Efficient Infection of Epithelial Cells with Human Adenovirus Type 26. J Virol. 2018 Dec 10 ; 93(1). pii: e01474-18.

human adenovirus type 26, epithelial cells, αvβ3 integrin, clathrin, caveolin

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