title: Plasmid-based human norovirus reverse genetics system produces reporter-tagged progeny virus containing infectious genomic RNA.
abstract: Human norovirus (HuNoV) is the leading cause of gastroenteritis worldwide. HuNoV replication studies have been hampered by the inability to grow the virus in cultured cells. The HuNoV genome is a positive-sense single-stranded RNA (ssRNA) molecule with three open reading frames (ORFs). We established a reverse genetics system driven by a mammalian promoter that functions without helper virus. The complete genome of the HuNoV genogroup II.3 U201 strain was cloned downstream of an elongation factor-1α (EF-1α) mammalian promoter. Cells transfected with plasmid containing the full-length genome (pHuNoVU201F) expressed the ORF1 polyprotein, which was cleaved by the viral protease to produce the mature nonstructural viral proteins, and the capsid proteins. Progeny virus produced from the transfected cells contained the complete NoV genomic RNA (VP1, VP2, and VPg) and exhibited the same density in isopycnic cesium chloride gradients as native infectious NoV particles from a patient's stool. This system also was applied to drive murine NoV RNA replication and produced infectious progeny virions. A GFP reporter construct containing the GFP gene in ORF1 produced complete virions that contain VPg-linked RNA. RNA from virions containing the encapsidated GFP-genomic RNA was successfully transfected back into cells producing fluorescent puncta, indicating that the encapsidated RNA is replication-competent. The EF-1α mammalian promoter expression system provides the first reverse genetics system, to our knowledge, generalizable for human and animal NoVs that does not require a helper virus. Establishing a complete reverse genetics system expressed from cDNA for HuNoVs now allows the manipulation of the viral genome and production of reporter virions.
authors.affiliation: Departments of Molecular Virology and Microbiology and Department of Virology II, National Institute of Infectious Diseases, Tokyo 208-0011, Japan; and.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Katayama
authors.affiliation: Departments of Molecular Virology and Microbiology and Department of Virology II, National Institute of Infectious Diseases, Tokyo 208-0011, Japan; and.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Murakami
authors.affiliation: Departments of Molecular Virology and Microbiology and.
authors.forename: Tyler M
authors.initials: TM
authors.lastname: Sharp
authors.affiliation: Departments of Molecular Virology and Microbiology and.
authors.forename: Susana
authors.initials: S
authors.lastname: Guix
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo 208-0011, Japan; and.
authors.forename: Tomoichiro
authors.initials: T
authors.lastname: Oka
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo 208-0011, Japan; and.
authors.forename: Reiko
authors.initials: R
authors.lastname: Takai-Todaka
authors.affiliation: Section of Gene Therapy, Department of Aging Intervention, National Center for Geriatrics and Gerontology, Aichi 474-8511, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Nakanishi
authors.affiliation: Departments of Molecular Virology and Microbiology and.
authors.forename: Sue E
authors.initials: SE
authors.lastname: Crawford
authors.affiliation: Departments of Molecular Virology and Microbiology and Medicine, Baylor College of Medicine, Houston, TX 77030;
authors.forename: Robert L
authors.initials: RL
authors.lastname: Atmar
authors.affiliation: Departments of Molecular Virology and Microbiology and Medicine, Baylor College of Medicine, Houston, TX 77030; mestes@bcm.edu.
authors.forename: Mary K
authors.initials: MK
authors.lastname: Estes
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics; D017930:Genes, Reporter; D016679:Genome, Viral; D006801:Humans; D029322:Norovirus / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames / Q000502:physiology; D020648:Peptide Elongation Factor 1 / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics; D014771:Virion / Q000235:genetics / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D020648:Peptide Elongation Factor 1; D012367:RNA, Viral; D014764:Viral Proteins
keywords: helper-virus–free reverse genetic system; reporter-tagged norovirus