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

Nora Mcfadden,Peter Simmonds,Felix Yarovinsky,Guia Carrara,Andrew Macdonald,Dalan Bailey,Amita Shortland,Ian Goodfellow,Yasmin Chaudhry,Jonathan Heeney,Alicia Benson

doi:10.1371/journal.ppat.1002413

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.

Highlights

The innate and adaptive immune systems result in a strong evolutionary pressure on pathogens to develop countermeasures to allow their continued existence
Unlike other members of the Caliciviridae, which typically encode three open reading frames [6], our analysis and that presented during large scale sequencing of many murine norovirus (MNV) genomes [32] indicates the presence of a fourth potential ORF in the MNV genome (Figure 1A) In this study we demonstrate that the protein encoded by ORF4 is expressed during virus infection, is not essential for virus replication in tissue culture but plays a role in viral virulence and represents a novel viral virulence factor
Bioinformatic prediction and analysis of ORF4/virulence factor 1 (VF1) The region of the genome encoding VF1 contains an intact reading frame in all available MNV sequences derived from different isolates or strains (Figure 1A, 1B and data not shown)

Summary

Introduction

The innate and adaptive immune systems result in a strong evolutionary pressure on pathogens to develop countermeasures to allow their continued existence. As a likely direct consequence, RNA viruses have evolved a variety of mechanisms to increase the coding capacity of their genomes [2]. These include the use of ribosomal frameshifting where a proportion of translating ribosomes change the reading frame to produce proteins with common N-terminal but a different C-. Many viruses have evolved to use a mechanism that creates overlapping reading frames through the use of two or more transcription initiation sites or translation start codons within the same RNA sequence [4,5]

Methods

Results

Conclusion