Integrin - Mediated Entry of Echovirus 1

Upla, Paula Integrin-mediated entry of echovirus 1 Jyväskylä: University of Jyväskylä, 2008, 86 p. (Jyväskylä Studies in Biological and Environmental Science, ISSN 1456-9701; 189) ISBN 978-951-39-3109-4 (PDF), 978-951-39-3087-5 Yhteenveto: Echovirus 1:n integriinivälitteinen sisäänmeno soluun Diss. Echovirus 1 (EV1) is a human pathogen, which belongs to the Picornaviridae family of RNA viruses. Here, the life cycle of EV1 was analyzed in detail. Binding of EV1 to the cell surface α2β1 integrin, a collagen receptor, was not accompanied by a conformational change in the virus capsid needed for the release of the RNA genome. EV1 pentamer is supposed to bind five integrin heterodimers at the same time and, behaving as a multivalent ligand, to cause integrin clustering. Clustering of integrins is important for signaling events. Formation of integrin clusters was initiated using primary anti-α2 integrin and secondary antibodies. The receptor clusters were seen to move laterally from glycosyl phosphatidylinositol anchored protein (GPI-AP) enriched domains along cortical actin filaments and to subsequently internalize. The internalization process was dependent on protein kinase Cα (PKCα) activity and other signaling events. Integrin α2β1 clustered by antibodies or EV1 was internalized into perinuclearly accumulated vesicles positive for caveolin-1, but not in endosomal vesicles of the clathrin-dependent entry route. The perinuclear vesicles were caveosomes, since EV1 partially colocalized there with simian virus 40 (SV40) and cholera toxin, known to traffic via caveosomes. However, colocalization of EV1 with caveolin-1 was not evident at the plasma membrane, but became apparent during the first 10 to 20 min of internalization. EV1 did not move to any other cellular locations from caveosomes. Presumably the EV1 RNA genome is released directly from the caveosomes to initiate replication in the cytoplasm. The replication of EV1 was dependent on cytosolic Ca2+-activated cysteine proteases, calpains, which degrade a number of cytoskeletal and cytoplasmic proteins. Both calpains 1 and 2 were recruited into caveosomes and they were activated concomitantly with the increase in Ca2+ concentration. Calpain inhibitors, as well as siRNAs for calpains 1 and 2 blocked EV1 infection. However, calpain inhibition did not have preventive effect on the in vitro translation of viral proteins.