title: Norwalk virus RNA is infectious in mammalian cells.
abstract: Human noroviruses are positive-sense RNA viruses and are the leading cause of epidemic acute viral gastroenteritis in developed countries. The absence of an in vitro cell culture model for human norovirus infection has limited the development of effective antivirals and vaccines. Human histo-blood group antigens have been regarded as receptors for norovirus infection, and expression of the alpha(1,2) fucosyltransferase gene (FUT2) responsible for the secretor phenotype is required for susceptibility to Norwalk virus (NV) infection. We report for the first time that transfection of NV RNA, isolated from stool samples from human volunteers, into human hepatoma Huh-7 cells leads to viral replication, with expression of viral antigens, RNA replication, and release of viral particles into the medium. Prior treatment of the RNA with proteinase K completely abolishes RNA infectivity, suggesting a key role of an RNA-protein complex. Although overexpression of the human FUT2 gene enhances virus binding to cells, it is not sufficient to allow a complete viral infection, and viral spread from NV-transfected cells to naïve cells does not occur. Finally, no differences in NV RNA replication are observed between Huh-7 and Huh-7.5.1 cells, which contain an inactivating mutation in retinoic acid-inducible gene I (RIG-I), suggesting that the RIG-I pathway does not play a role in limiting NV replication. Our results strongly suggest that the block(s) to NV replication in vitro is at the stage of receptor and/or coreceptor binding and/or uncoating, either because cells lack some specific factor or activation of cellular antiviral responses independent of RIG-I inhibits virus replication.
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza BCM-385, Houston, TX 77030, USA.
authors.forename: Susana
authors.initials: S
authors.lastname: Guix
authors.forename: Miyuki
authors.initials: M
authors.lastname: Asanaka
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Katayama
authors.forename: Sue E
authors.initials: SE
authors.lastname: Crawford
authors.forename: Frederick H
authors.initials: FH
authors.lastname: Neill
authors.forename: Robert L
authors.initials: RL
authors.lastname: Atmar
authors.forename: Mary K
authors.initials: MK
authors.lastname: Estes
mesh_terms: D000956:Antigens, Viral / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D005243:Feces / Q000821:virology; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism; D016679:Genome, Viral; D006801:Humans; D009663:Norwalk virus / Q000235:genetics / Q000502:physiology*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011971:Receptors, Immunologic; D014162:Transfection; D014779:Virus Replication; D000097631:Galactoside 2-alpha-L-fucosyltransferase
chemical_list: D000956:Antigens, Viral; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases; D005647:Fucosyltransferases; D012367:RNA, Viral; D011971:Receptors, Immunologic; D000097631:Galactoside 2-alpha-L-fucosyltransferase; C452820:RIGI protein, human