title: Integrins are not essential for entry of coxsackievirus A9 into SW480 human colon adenocarcinoma cells.
abstract: Coxsackievirus A9 (CV-A9) is a pathogenic enterovirus type within the family Picornaviridae. CV-A9 infects A549 human epithelial lung carcinoma cells by attaching to the αVβ6 integrin receptor through a highly conserved Arg-Gly-Asp (RGD) motif, which is located at the exposed carboxy-terminus of the capsid protein VP1 detected in all studied clinical isolates. However, genetically-modified CV-A9 that lacks the RGD motif (CV-A9-RGDdel) has been shown to be infectious in some cell lines but not in A549, suggesting that RGD-mediated integrin binding is not always essential for efficient entry of CV-A9.
  
  
  
  Two cell lines, A549 and SW480, were used in the study. SW480 was the study object for the integrin-independent entry and A549 was used as the control for integrin-dependent entry. Receptor levels were quantitated by cell sorting and quantitative PCR. Antibody blocking assay and siRNA silencing of receptor-encoding genes were used to block virus infection. Peptide phage display library was used to identify peptide binders to CV-A9. Immunofluorescence and confocal microscopy were used to visualize the virus infection in the cells.
  
  
  
  We investigated the receptor use and early stages of CV-A9 internalization to SW480 human epithelial colon adenocarcinoma cells. Contrary to A549 infection, we showed that both CV-A9 and CV-A9-RGDdel internalized into SW480 cells and that function-blocking anti-αV integrin antibodies had no effect on the binding and entry of CV-A9. Whereas siRNA silencing of β6 integrin subunit had no influence on virus infection in SW480, silencing of β2-microglobulin (β2M) inhibited the virus infection in both cell lines. By using a peptide phage display screening, the virus-binding peptide identical to the N-terminal sequence of HSPA5 protein was identified and shown to block the virus infection in both A549 and SW480 cell lines. HSPA5 was also found to co-localize with CV-A9 at the SW480 cell periphery during the early stages of infection by confocal microscopy.
  
  
  
  The data suggest that while αVβ6 integrin is essential for CV-A9 in A549 cell line, it is not required in SW480 cell line in which β2M and HSPA5 alone are sufficient for CV-A9 infection. This suggests that the choice of CV-A9 receptor(s) is dependent on the tissue/cellular environment.
authors.affiliation: Department of Virology, University of Turku, Turku, Finland.
authors.forename: Outi
authors.initials: O
authors.lastname: Heikkilä
authors.affiliation: Department of Virology, University of Turku, Turku, Finland.
authors.forename: Pirjo
authors.initials: P
authors.lastname: Merilahti
authors.affiliation: Department of Virology, University of Turku, Turku, Finland.
authors.forename: Marika
authors.initials: M
authors.lastname: Hakanen
authors.affiliation: Department of Virology, University of Turku, Turku, Finland.|Department of Medical Microbiology, Laboratory of Clinical Virology, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Eveliina
authors.initials: E
authors.lastname: Karelehto
authors.affiliation: Department of Virology, University of Turku, Turku, Finland.|Turku Centre for Biotechnology, University of Turku, Turku, Finland.
authors.forename: Jonna
authors.initials: J
authors.lastname: Alanko
authors.affiliation: Department of Virology, University of Turku, Turku, Finland.
authors.forename: Maria
authors.initials: M
authors.lastname: Sukki
authors.affiliation: Department of Virology, University of Turku, Turku, Finland.|Department of Bacteriology and Immunology, Research Programs Unit, Immunobiology, University of Helsinki, and Helsinki University Hospital, Helsinki, Finland.
authors.forename: Saija
authors.initials: S
authors.lastname: Kiljunen
authors.affiliation: Department of Virology, University of Turku, Turku, Finland. pesusi@utu.fi.|Biomaterials and Diagnostics Group, Turku University of Applied Sciences, Turku, Finland. pesusi@utu.fi.
authors.forename: Petri
authors.identifier: 0000-0002-9245-3271
authors.initials: P
authors.lastname: Susi
mesh_terms: D000951:Antigens, Neoplasm / Q000378:metabolism*; D045744:Cell Line, Tumor; D000091342:Endoplasmic Reticulum Chaperone BiP; D029822:Enterovirus B, Human / Q000502:physiology*; D004847:Epithelial Cells / Q000821:virology*; D054884:Host-Pathogen Interactions; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D011991:Receptors, Virus / Q000378:metabolism*; D053586:Virus Internalization
chemical_list: D000951:Antigens, Neoplasm; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D016023:Integrins; D011991:Receptors, Virus; C073878:integrin alphavbeta6
keywords: Coxsackievirus A9; HSPA5; Receptor; β2-microglobulin