Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4

PLoS Pathogens

Peter Simmonds¹  Andrew Macdonald²  Amita Shortland³  Jonathan Heeney³  Nora McFadden⁴  Ian Goodfellow⁴  Dalan Bailey⁴  Guia Carrara⁴  Yasmin Chaudhry⁴  Alicia Benson⁵  Felix Yarovinsky⁵ 

[1] Centre for Immunology, Infection and Evolution, University of Edinburgh, Edinburgh, United Kingdom;Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom;Laboratory of Viral Zoonotics, Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom;Section of Virology, Department of Medicine, Imperial College London, London, United Kingdom;University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America

关键词: Viral replication;    Mitochondria;    Apoptosis;    Immune response;    Norovirus;    Mouse models;    Tissue cultures;    RNA viruses;   

DOI  :  10.1371/journal.ppat.1002413

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY