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Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake
Oliver Meier1, Karin Boucke, Silvija Vig Hammer
1Zoologisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Abstract:
Adenovirus type 2 (Ad2) binds the coxsackie B virus Ad receptor and is endocytosed upon activation of the alphav 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 alphav 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.
Insights
Adenovirus (Ad) uses clathrin-dependent endocytosis and hijacks macropinocytosis, a fluid uptake process, to infect cells. This process involves signaling that induces macropinosome leakage, aiding viral escape into the cytosol.
Area of Science:
- Cell Biology
- Virology
- Molecular Biology
Background:
- Adenovirus (Ad) entry into epithelial cells is initiated by binding to the coxsackie B virus Ad receptor.
- Ad uptake is further facilitated by the activation of alphav integrin coreceptors.
Purpose of the Study:
- To investigate the role of clathrin-mediated endocytosis and macropinocytosis in Adenovirus type 2 (Ad2) infection.
- To elucidate the mechanisms by which Ad2 utilizes cellular endocytic pathways for entry and infection.
Main Methods:
- Expression of dominant-negative inhibitors (clathrin hub, eps15, K44A-dynamin) to block specific endocytic pathways.
- Monitoring fluid phase tracer uptake and transferrin uptake to assess endocytic activity.
- Investigating the dependence of macropinocytosis on specific cellular components (alphav integrins, PKC, F-actin, Na+/H+ exchanger).
Main Results:
- Ad2 uptake was inhibited by blocking clathrin-dependent endocytosis.
- Ad2 significantly stimulated macropinocytosis, a fluid uptake process, independent of transferrin uptake.
- Macropinocytosis stimulation and subsequent macropinosomal lysis, releasing contents into the cytosol, were dependent on alphav integrins and other signaling molecules.
- Macropinosomal lysis occurred even when viral escape was blocked, indicating Ad-induced signaling controls this process.
Conclusions:
- Adenovirus type 2 utilizes clathrin-dependent endocytosis for cellular entry.
- Ad2 actively induces and exploits macropinocytosis, leading to macropinosome leakage and release of contents into the cytosol.
- Cell surface signaling by Ad controls macropinosome formation and lysis, correlating with viral escape and infection.