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Role of clathrin and caveolin in human adenovirus type 26 cell entry

PURPOSE OF WORK. Low-prevalence adenoviruses such as human adenovirus type 26 (HAdV26) may allow the development of a more efficient and safer viral vector. Recently we reported that αvβ3 integrin is required for efficient infection of epithelial cells with HAdV26, however detailed insight into the mechanisms of HAdV26 entry is still missing. Here, we investigated the influence of dynamin-2, clathrin and caveolin-1 on HAdV26 endocytosis in A549 cell line. MATERIALS AND METHODS. The knockdown of dynamin-2, clathrin and caveolin-1 was achieved by transient transfection of siRNA or stable transfection of shRNA. Flow cytometry and confocal microscopy were used to study adenovirus transduction efficiency and internalization, and western blot for protein expression. RESULTS. Dynamin-2 is involved in HAdV26 infection as its downregulation or inhibition decreased transduction efficiency or internalization of HAdV26. Decreased expression of clathrin strongly increased HAdV26 transduction efficiency and internalization of HAdV26 in A549. Clathrin downregulation increased the expression of αvβ3 integrin on the cell surface which could explain the increased HAdV26 entry. Additionally, a significant increase in HAdV26 transduction efficiency was obtained by using pitstop 2, a selective inhibitor of clathrin-mediated endocytosis. The use of pitstop 2 blocked the internalization of HAdV26, stopping viruses near the membrane. After recovery in medium without pitstop 2, HAdV26 successfully trafficked within the cell, with significantly higher number of viral particles in pitstop 2-treated compared to untreated samples. Although decreased expression of caveolin-1 did not significantly affect HAdV26 infection in A549, downregulation of caveolin-1 in clones A549-E6 which has higher expression of αvβ3 integrin, and A549-69 which has decreased expression of clathrin and higher expression of αvβ3 integrin, decreased HAdV26 entry indicating that the αvβ3 integrin directs HAdV26 to caveolin- 1-mediated endocytosis. CONCLUSION. Based on our results we conclude that HAdV26 endocytosis is both clathrin- and caveolin-1- dependent, whereby role of clathrin is evident in two steps of HAdV26 cell entry: i) altering amount of αvβ3 integrin on cell surface, and ii) blocking adenoviral particles in the close proximity of cell membrane, while caveolin-1 has role in cells with high expression of αvβ3 integrin. 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. 2019 Jan 1 ; 93(1): e01474-18.

human adenovirus type 26 ; cell entry ; clathrin ; caveolin

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