title: Echovirus 1 endocytosis into caveosomes requires lipid rafts, dynamin II, and signaling events.
abstract: Binding of echovirus 1 (EV1, a nonenveloped RNA virus) to the alpha2beta1 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.
authors.affiliation: Department of Virology, Haartman Institute, University of Helsinki, FIN-00014 Helsinki, Finland. vilja.pietiainen@helsinki.fi
authors.forename: Vilja
authors.initials: V
authors.lastname: Pietiäinen
authors.forename: Varpu
authors.initials: V
authors.lastname: Marjomäki
authors.forename: Paula
authors.initials: P
authors.lastname: Upla
authors.forename: Lucas
authors.initials: L
authors.lastname: Pelkmans
authors.forename: Ari
authors.initials: A
authors.lastname: Helenius
authors.forename: Timo
authors.initials: T
authors.lastname: Hyypiä
mesh_terms: D000818:Animals; D001665:Binding Sites; D020126:Brefeldin A / Q000494:pharmacology; D002213:Capsid / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002772:Cholera Toxin / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D034285:Dynamin II / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D029822:Enterovirus B, Human / Q000378:metabolism*; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015151:Immunoblotting; D017403:In Situ Hybridization; D017404:In Situ Hybridization, Fluorescence; D007700:Kinetics; D021962:Membrane Microdomains / Q000378:metabolism* / Q000821:virology; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D010766:Phosphorylation; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism; D015398:Signal Transduction; D013395:Sucrose / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
chemical_list: D012367:RNA, Viral; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; D013395:Sucrose; D002772:Cholera Toxin; D002784:Cholesterol; D034285:Dynamin II