Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4 Nora McFadden 1. , Dalan Bailey 1. , Guia Carrara 1¤ , Alicia Benson 2 , Yasmin Chaudhry 1 , Amita Shortland 3 , Jonathan Heeney 3 , Felix Yarovinsky 2 , Peter Simmonds 4 , Andrew Macdonald 5 , Ian Goodfellow 1 * 1 Section of Virology, Department of Medicine, Imperial College London, London, United Kingdom, 2 University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America, 3 Laboratory of Viral Zoonotics, Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom, 4 Centre for Immunology, Infection and Evolution, University of Edinburgh, Edinburgh, United Kingdom, 5 Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom 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. Citation: McFadden N, Bailey D, Carrara G, Benson A, Chaudhry Y, et al. (2011) Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4. PLoS Pathog 7(12): e1002413. doi:10.1371/journal.ppat.1002413 Editor: Christopher F. Basler, Mount Sinai School of Medicine, United States of America Received May 2, 2011; Accepted October 12, 2011; Published December 8, 2011 Copyright: ß 2011 McFadden et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: IG is a Wellcome Trust Senior fellow. This work was supported by a Wellcome Trust Senior Fellowship to IG (WT081624MA) as well as funding from the Society for General Microbiology and Imperial College London. AM is an RCUK Academic Fellow and research in his group is funded by Yorkshire Cancer Research (LPP041, PP015 and L33) and the Royal Society. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: I.Goodfellow@Imperial.ac.uk ¤ Current address: Department of Pathology, University of Cambridge, Cambridge, United Kingdom . These authors contributed equally to this work. Introduction Collectively, the innate and adaptive immune systems result in a strong evolutionary pressure on pathogens to develop counter- measures to allow their continued existence. Therefore pathogens, including viruses, have evolved a multitude of mechanisms for evading the host response to infection, often by the expression of proteins that interfere with cellular antimicrobial response mechanisms [1]. The size of RNA virus genomes is thought to be limited by the error prone nature of the viral polymerase. 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- terminal from the read-through sequence [3]. Many viruses have also 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]. Murine norovirus (MNV) was identified in 2003 as a virus that caused a lethal infection in immunocompromised mice [6]. However, MNV is now known to be a widespread infectious agent of laboratory mice with a reported seroprevalence of 20- 64% [7,8]. MNV is currently the only norovirus which replicates efficiently in tissue culture, where it has a tropism for dendritic and macrophage cells [9]. The availability of immortalized macro- phage cell lines such as the murine macrophage RAW264.7, has allowed significant advances to be made in understanding the life cycle of this virus. For the first time critical processes in the norovirus life cycle have been dissected e.g. the mechanism of PLoS Pathogens | www.plospathogens.org 1 December 2011 | Volume 7 | Issue 12 | e1002413