title: Norovirus regulation of the innate immune response and apoptosis occurs via the product of the alternative open reading frame 4.
abstract: Small RNA viruses have evolved many mechanisms to increase the capacity of their short genomes. Here we describe the identification and characterization of a novel open reading frame (ORF4) encoded by the murine norovirus (MNV) subgenomic RNA, in an alternative reading frame overlapping the VP1 coding region. ORF4 is translated during virus infection and the resultant protein localizes predominantly to the mitochondria. Using reverse genetics we demonstrated that expression of ORF4 is not required for virus replication in tissue culture but its loss results in a fitness cost since viruses lacking the ability to express ORF4 restore expression upon repeated passage in tissue culture. Functional analysis indicated that the protein produced from ORF4 antagonizes the innate immune response to infection by delaying the upregulation of a number of cellular genes activated by the innate pathway, including IFN-Beta. Apoptosis in the RAW264.7 macrophage cell line was also increased during virus infection in the absence of ORF4 expression. In vivo analysis of the WT and mutant virus lacking the ability to express ORF4 demonstrated an important role for ORF4 expression in infection and virulence. STAT1-/- mice infected with a virus lacking the ability to express ORF4 showed a delay in the onset of clinical signs when compared to mice infected with WT virus. Quantitative PCR and histopathological analysis of samples from these infected mice demonstrated that infection with a virus not expressing ORF4 results in a delayed infection in this system. In light of these findings we propose the name virulence factor 1, VF1 for this protein. The identification of VF1 represents the first characterization of an alternative open reading frame protein for the calicivirus family. The immune regulatory function of the MNV VF1 protein provide important perspectives for future research into norovirus biology and pathogenesis.
authors.affiliation: Section of Virology, Department of Medicine, Imperial College London, London, United Kingdom.
authors.forename: Nora
authors.initials: N
authors.lastname: McFadden
authors.forename: Dalan
authors.initials: D
authors.lastname: Bailey
authors.forename: Guia
authors.initials: G
authors.lastname: Carrara
authors.forename: Alicia
authors.initials: A
authors.lastname: Benson
authors.forename: Yasmin
authors.initials: Y
authors.lastname: Chaudhry
authors.forename: Amita
authors.initials: A
authors.lastname: Shortland
authors.forename: Jonathan
authors.initials: J
authors.lastname: Heeney
authors.forename: Felix
authors.initials: F
authors.lastname: Yarovinsky
authors.forename: Peter
authors.initials: P
authors.lastname: Simmonds
authors.forename: Andrew
authors.initials: A
authors.lastname: Macdonald
authors.forename: Ian
authors.initials: I
authors.lastname: Goodfellow
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000235:genetics* / Q000276:immunology; D015153:Blotting, Western; D017250:Caliciviridae Infections / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D007113:Immunity, Innate / Q000235:genetics*; D047468:Immunoprecipitation; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D029322:Norovirus / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity; D016366:Open Reading Frames; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D059386:Reverse Genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014764:Viral Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D014774:Virulence / Q000235:genetics; D037521:Virulence Factors / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics
chemical_list: D012367:RNA, Viral; D014764:Viral Proteins; D037521:Virulence Factors