Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake.
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.
Meier
Oliver
O
Zoologisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Boucke
Karin
K
Hammer
Silvija Vig
SV
Keller
Stephan
S
Stidwill
Robert P
RP
Hemmi
Silvio
S
Greber
Urs F
UF
D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000256:Adenoviridae / Q000378:metabolism* / Q000472:pathogenicity* / Q000648:ultrastructure; D000258:Adenovirus Infections, Human / Q000821:virology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002966:Clathrin / Q000502:physiology*; D003034:Coated Pits, Cell-Membrane / Q000648:ultrastructure; D018699:Coated Vesicles / Q000648:ultrastructure; D003600:Cytosol / Q000821:virology; D034281:Dynamins; D011992:Endosomes / Q000378:metabolism*; D004847:Epithelial Cells / Q000821:virology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D016023:Integrins / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins / Q000378:metabolism; D010873:Pinocytosis; D011493:Protein Kinase C / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000502:physiology; D014168:Transferrin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D002966:Clathrin; C486206:EPS15 protein, human; D016023:Integrins; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; D014168:Transferrin; D011493:Protein Kinase C; D020558:GTP Phosphohydrolases; D034281:Dynamins