Opening of size-selective pores in endosomes during human rhinovirus serotype 2 in vivo uncoating monitored by single-organelle flow analysis.
The effect of virus uncoating on endosome integrity during the early steps in viral infection was investigated. Using fluid-phase uptake of 10- and 70-kDa dextrans labeled with a pH-dependent fluorophore (fluorescein isothiocyanate [FITC]) and a pH-independent fluorophore (cyanine 5 [Cy5]), we determined the pHs of labeled compartments in intact HeLa cells by fluorescence-activated cell sorting analysis. Subsequently, the number and pH of fluorescent endosomes in cell homogenates were determined by single-organelle flow analysis. Cointernalization of adenovirus and 70-kDa FITC- and Cy5-labeled dextran (FITC/Cy5-dextran) led to virus-induced endosomal rupture, resulting in the release of the marker from the low-pH environment into the neutral cytosol. Consequently, in the presence of adenovirus, the number of fluorescent endosomes was reduced by 40% compared to that in the control. When human rhinovirus serotype 2 (HRV2) was cointernalized with 10-and 70-kDa FITC/Cy5-dextrans, the 10-kDa dextran was released, whereas the 70-kDa dextran remained within the endosomes, which also maintained their low pH. These data demonstrate that pores are generated in the membrane during HRV2 uncoating and RNA penetration into the cytosol without gross damage of the endosomes; 10-kDa dextran can access the cytosol through these pores. Whereas rhinovirus-mediated pore formation was prevented by the vacuolar ATPase inhibitor bafilomycin A1, adenovirus-mediated endosomal rupture also occurred in the presence of the inhibitor. This finding is in keeping with the low-pH requirement of HRV2 infection; for adenovirus, no pH dependence for endosomal escape was found with this drug.
Brabec
Marianne
M
Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria.
Schober
Daniela
D
Wagner
Ernst
E
Bayer
Nora
N
Murphy
Robert F
RF
Blaas
Dieter
D
Fuchs
Renate
R
D000256:Adenoviridae / Q000502:physiology; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D012367:RNA, Viral / Q000378:metabolism; D012229:Rhinovirus / Q000145:classification / Q000502:physiology*; D012703:Serotyping
D012367:RNA, Viral