title: Attachment and entry of recombinant Norwalk virus capsids to cultured human and animal cell lines.
abstract: Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant Norwalk virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. We have used these rNV VLPs to examine virus-cell interactions. Binding and internalization of VLPs to cultured human and animal cell lines were studied in an attempt to identify potentially susceptible cell lines for virus propagation in vitro and to determine if early events in the replication cycle were responsible for the narrow host range and restriction of virus growth in cell culture. Radiolabeled VLPs specifically bound to a saturable number of binding molecules on the cell surface of 13 cell lines from different origins, including human intestine (differentiated and undifferentiated Caco-2) and insect (Spodoptera frugiperda 9) ovary. Differentiated Caco-2 cells bound significantly more rNV VLPs than the other cell lines. Variations in the amount of bound VLPs among the different cell lines did not correlate with the tissue or species of origin. VLP binding was specific, as determined by competition experiments with unlabeled rNV VLPs; however, only 1.4 to 6.8% of the specifically prebound radiolabeled VLPs became internalized into cells. Blocking experiments using polygonal and monoclonal anti-rNV sera and specific antipeptide sera were performed to map the domains on rNV VLPs involved in binding to cells. One monoclonal antibody (NV8812) blocked binding of rNV VLPs to human and animal cell lines. The binding site of monoclonal antibody NV8812 was localized to the C-terminal 300 to 384 residues of the capsid protein by immunoprecipitation with truncated and cleaved forms of the capsid protein. These data suggest that the C-terminal region of the capsid protein is involved in specific binding of rNV VLPs to cells.
authors.affiliation: Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: L J
authors.initials: LJ
authors.lastname: White
authors.forename: J M
authors.initials: JM
authors.lastname: Ball
authors.forename: M E
authors.initials: ME
authors.lastname: Hardy
authors.forename: T N
authors.initials: TN
authors.lastname: Tanaka
authors.forename: N
authors.initials: N
authors.lastname: Kitamoto
authors.forename: M K
authors.initials: MK
authors.lastname: Estes
mesh_terms: D000818:Animals; D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D009663:Norwalk virus / Q000502:physiology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
chemical_list: D011994:Recombinant Proteins