title: Recovery of infectious murine norovirus using pol II-driven expression of full-length cDNA.
abstract: Noroviruses are the major cause of nonbacterial gastroenteritis in humans. These viruses have remained refractory to detailed molecular studies because of the lack of a reverse genetics system coupled to a permissive cell line for targeted genetic manipulation. There is no permissive cell line in which to grow infectious human noroviruses nor an authentic animal model that supports their replication. In contrast, murine norovirus (MNV) offers a tractable system for the study of noroviruses with the recent discovery of permissive cells and a mouse model. The lack of a reverse genetic system for MNV has been a significant block to understanding the biology of noroviruses. We report recovery of infectious MNV after baculovirus delivery of viral cDNA to human hepatoma cells under the control of an inducible DNA polymerase (pol) II promoter. Recovered virus replicated in murine macrophage (RAW264.7) cells, and the recovery of MNV from DNA was confirmed through recovery of virus containing a marker mutation. This pol II promoter driven expression of viral cDNA also generated infectious virus after transfection of HEK293T cells, thus providing both transduction and transfection systems for norovirus reverse genetics. We used norovirus reverse genetics to demonstrate by mutagenesis of the protease-polymerase (pro-pol) cleavage site that processing of pro-pol is essential for the recovery of infectious MNV. This represents the first infectious reverse genetics system for a norovirus, and should provide approaches to address fundamental questions in norovirus molecular biology and replication.
authors.affiliation: Department of Microbiology and Immunology, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand. vernon.ward@stonebow.otago.ac.nz
authors.forename: Vernon K
authors.initials: VK
authors.lastname: Ward
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: McCormick
authors.forename: Ian N
authors.initials: IN
authors.lastname: Clarke
authors.forename: Omar
authors.initials: O
authors.lastname: Salim
authors.forename: Christiane E
authors.initials: CE
authors.lastname: Wobus
authors.forename: Larissa B
authors.initials: LB
authors.lastname: Thackray
authors.forename: Herbert W
authors.initials: HW
authors.lastname: Virgin
authors.forename: Paul R
authors.initials: PR
authors.lastname: Lambden
mesh_terms: D000818:Animals; D017250:Caliciviridae Infections / Q000821:virology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D045744:Cell Line, Tumor; D004257:DNA Polymerase II / Q000502:physiology*; D018076:DNA, Complementary; D015870:Gene Expression; D005814:Genes, Viral; D006801:Humans; D051379:Mice; D029322:Norovirus / Q000235:genetics / Q000302:isolation & purification*; D011499:Protein Processing, Post-Translational; D014161:Transduction, Genetic; D014162:Transfection; D014779:Virus Replication / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D004257:DNA Polymerase II