title: Investigation of norovirus replication in a human cell line.
abstract: Noroviruses (NoVs) belong to the genus Norovirus and are members of the family Caliciviridae. NoVs are the dominant cause of outbreaks of gastroenteritis, but progress in understanding the molecular characteristics of NoV and its replication strategies have been hampered by the lack of a cell culture system or a practical animal model, except for murine NoVs. To elucidate the transcription and replication of the NoV genome, a complete genome of a human NoV genogroup II strain was cloned downstream of a T7 RNA polymerase promoter and expressed in human embryonic kidney (HEK) 293T/17 cells using a T7 vaccinia virus expression system. Bands for a 7.6-kb negative-strand RNA, a 7.6-kb positive-strand genomic RNA, and a 2.6-kb positive-strand subgenomic-like RNA were found in the infected cells. However, recombinant capsid protein (rVP1) and rVP2 were not detected by Western blotting. When a construct containing VP1 and VP2 genes was co-transfected with a full-length construct, the expression of virus-like particles (VLPs) with a buoyant density of 1.271 g/cm3 was observed. We also observed round particles, 20 to 80 nm in diameter, with a buoyant density of 1.318 g/cm3. Our results indicated that NoV RNA was incorporated into the heavier particles. However, further studies are needed to investigate the antigenicity of these particles and to determine if they represent undeveloped VLPs.
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Katayama
authors.forename: G S
authors.initials: GS
authors.lastname: Hansman
authors.forename: T
authors.initials: T
authors.lastname: Oka
authors.forename: S
authors.initials: S
authors.lastname: Ogawa
authors.forename: N
authors.initials: N
authors.lastname: Takeda
mesh_terms: D015152:Blotting, Northern; D015153:Blotting, Western; D036022:Capsid Proteins / Q000032:analysis; D002460:Cell Line; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D016679:Genome, Viral; D006801:Humans; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D029322:Norovirus / Q000235:genetics / Q000502:physiology*; D012367:RNA, Viral / Q000032:analysis; D014616:Vaccinia virus; D014764:Viral Proteins / Q000235:genetics; D022701:Virosomes / Q000302:isolation & purification / Q000648:ultrastructure; D014779:Virus Replication
chemical_list: D036022:Capsid Proteins; D012367:RNA, Viral; D014764:Viral Proteins; D022701:Virosomes; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases