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Vol. 15, Issue 11, 4911-4925, November 2004
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* Department of Virology, Haartman Institute, University of Helsinki, FIN-00014 Helsinki, Finland;
Department of Biological and Environmental Science, University of Jyväskylä, FIN-40351 Jyväskylä, Finland;
Max Planck Institute for Molecular Cell Biology and Genetics, D-01307 Dresden, Germany;
|| Swiss Federal Institute of Technology Zürich (ETH), CH-8093 Zürich, Switzerland; and
¶ Department of Virology and MediCity Research Laboratory, University of Turku, FIN-20520 Turku, Finland
Submitted January 29, 2004;
Revised August 17, 2004;
Accepted August 24, 2004
Monitoring Editor: Jean Gruenberg
Binding of echovirus 1 (EV1, a nonenveloped RNA virus) to the 
2
1 integrin on the cell surface is followed by endocytic internalization of the virus together with the receptor. Here, video-enhanced live microscopy revealed the rapid uptake of fluorescently labeled EV1 into mobile, intracellular structures, positive for green fluorescent protein-tagged caveolin-1. Partial colocalization of EV1 with SV40 (SV40) and cholera toxin, known to traffic via caveosomes, demonstrated that the vesicles were caveosomes. The initiation of EV1 infection was dependent on dynamin II, cholesterol, and protein phosphorylation events. Brefeldin A, a drug that prevents SV40 transport, blocked the EV1 infection cycle, whereas drugs that disrupt the cellular cytoskeleton had no effect. In situ hybridization revealed the localization of viral RNA with endocytosed viral capsid proteins in caveosomes before initiation of viral replication. Thus, both the internalization of EV1 to caveosomes and subsequent events differ clearly from caveolar endocytosis of SV40 because EV1 uptake is fast and independent of actin and EV1 is not sorted further to sER from caveosomes. These results shed further light on the cell entry of nonenveloped viral pathogens and illustrate the use of viruses as probes to dissect caveolin-associated endocytic pathways.
Abbreviations used: AF, Alexa Fluor-594; BFA, brefeldin A; Bis, bisindolylmaleimide; CTX, cholera toxin; Cy5, cyan 5 dye; CytD, cytochalasin D; EV1, echovirus 1; FISH, fluorescent in situ hybridization; Gen, genistein; GFP, green fluorescent protein; HRV, human rhinovirus; Jas, jasplakinolide; LatA, latrunculin A; MBCD, methyl--cyclodextrin; NaOV, sodium orthovanadate (Na3VO4); Noco, nocodazole; Nys, nystatin; OA, okadaic acid; p.i., postinfection (indicates time after 1-h incubation of EV1 at 4°C); PFU, plaque-forming unit; Prog, progesterone; PV, poliovirus; Saf, safingol; SV40, simian virus 40; TxR, Texas Red.
The online version of this article contains supplementary material accessible through http://www.molbiolcell.org.
Corresponding author. E-mail address: vilja.pietiainen{at}helsinki.fi.
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