Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake

Abstract

Adenovirus type 2 (Ad2) binds the coxsackie B virus Ad receptor and is endocytosed upon activation of the αv integrin coreceptors. Here, we demonstrate that expression of dominant negative clathrin hub, eps15, or K44A-dynamin (dyn) inhibited Ad2 uptake into epithelial cells, indicating clathrin-dependent viral endocytosis. Surprisingly, Ad strongly stimulated the endocytic uptake of fluid phase tracers, coincident with virus internalization but without affecting receptor-mediated transferrin uptake. A large amount of the stimulated endocytic activity was macropinocytosis. Macropinocytosis depended on αv integrins, PKC, F-actin, and the amiloride-sensitive Na+/H+ exchanger, which are all required for Ad escape from endosomes and infection. Macropinocytosis stimulation was not a consequence of viral escape, since it occurred in K44A-dyn–expressing cells. Surprisingly, 30–50% of the endosomal contents were released into the cytosol of control and also K44A-dyn–expressing cells, and the number of fluid phase–positive endosomes dropped below the levels of noninfected cells, indicating macropinosomal lysis. The release of macropinosomal contents was Ad dose dependent, but the presence of Ad particles on macropinosomal membranes was not sufficient for contents release. We conclude that Ad signaling from the cell surface controls the induction of macropinosome formation and leakage, and this correlates with viral exit to the cytosol and infection.

Virus trafficking – learning from single-virus tracking

“…Other viruses have also been observed to co-localize with CCPs in live cells, including reovirus and adenovirus 30,31 . In common with Influenza, reovirus enters by the de novo formation of CCPs 30 .…”

Intracellular trafficking of nonviral vectors

“…Studies on Ad-host interactions found that cytosolic release of macropinosomal contents may be triggered by receptor signaling. 91 While other types of cell entry pathways exist, some of which are little understood, those just described earlier have been identified as largely contributing to the uptake of numerous types of nonviral vectors. To delineate these cell entry pathways for the uptake of a particular vector, trafficking inhibitors, in addition to trafficking markers and special cell lines, can be used.…”

PCI-enhanced adenoviral transduction employs the known uptake mechanism of adenoviral particles

“…Other pathways have, however, been suggested, 40,41 and adenoviral particles have been observed in vesicles without clathrin coating and in macropinosomes. The enhanced gene transfer after photochemical treatment could be due to the release of the viral particles from any of these vesicles.…”