(A) The three previously described open reading frames (ORFs) are highlighted as ORF1, ORF2 and ORF3. The NS1-7 nomenclature of the mature peptides generated from ORF1 [as described by 86] is also shown. The position of the ORF4 reading frame is highlighted along with (B) the sequence of the 5′ end of the MNV sgRNA showing the start codons for VP1 and ORF4. (C) Synonymous and amino acid sequence variability scan of conventional MNV genes (ORF1, ORF2 and ORF3) with positions depicted to scale in genome diagram below axis. Position and variability of putative ORF4 superimposed on diagram. Log likelihood of alternate (ORF4) and null (ORFs 1, 2 and 3 only) coding predictions generated by MLOGD (mean values of 21 adjacent bases shown).

(A) Coupled in vitro transcription and translation of a cDNA construct containing the murine norovirus 1 (MNV) sgRNA was performed prior to immunoprecipitation (IP) with polyclonal antisera to VP1, VP2 or VF1. Immunoprecipitated complexes were subsequently resolved by SDS-PAGE (15% polyacrylamide) alongside an aliquot of the complete reaction prior to immunoprecipitation (SG). (B) Western blot analysis indicating VF1 expression during virus high multiplicity infection. RAW264.7 cells were infected with MNV-1 at a MOI of 5 TCID₅₀ per cell. Protein samples were prepared at various times post infections (HPI) and then separated by SDS-PAGE (15% polyacrylamide) prior to immune-blotting with antisera to VF1, NS7 or VP2.

(A) Schematic representation of the position and nucleotide changes introduced to generate the VF1 mutant viruses M1, M10 and M20. Nucleotide numbers refer to the positions in the MNV-1 genome. The effect of the introduced nucleotide changes on the VF1 and VP1 protein sequences are also illustrated. Note that the mutations introduced in M1, M10 and M20 do not affect the coding sequence of the capsid protein VP1 but introduce a stop codon into VF1 at various positions. (B) Coupled in vitro transcription and translation reactions confirming the lack of VF1 expression in M1 and M10 viruses, with M20 producing a marginally shorter VF1 product. PCR products containing the subgenomic RNA region under control of a T7 RNA polymerase promoter were generated from cDNA constructs of either wild-type (WT), M1, M10 or M20 VF1 mutants and subsequently used for transcription and translation (TNT) in vitro in the presence of S35 methionine. Radioactively labeled proteins were subsequent resolved by SDS-PAGE, prior to exposure to film. VP1* represents a potential shorter VP1 product generated by translation initiation from an AUG initiation codon in-frame yet downstream from the authentic VP1 AUG. (C) Western blot analysis of RAW264.7 cells infected with low passage, sequence verified stocks of either wild-type (WT), M1, M10 or M20 VF1 mutant viruses. RAW264.7 cells were infected at a MOI 10 TCID₅₀ per cell and harvested 12 hours post infection, prior to separation by SDS-PAGE and western blot using either anti-VF1 or anti-VP2 antisera. (D) Multi-cycle growth kinetics analysis of VF1 mutant viruses M1, M10 and M20 in RAW264.7 cells. Cells were infected with a MOI of 0.01 TCID₅₀ per cell and samples harvested at various times post infection prior to titration on RAW264.7 cells. Virus yield is expressed as TCID₅₀/ml. Infections were performed in triplicate, with the average virus titer and standard deviation plotted. (E) Sequence chromatograms of M1, M10 and M20 VF1 mutant viruses after passage 1 or 5 in RAW264.7 cells. Viruses obtained from passage 1 and 5 low multiplicity infections (MOI) of RAW264.7 cells were used to infect a subsequent monolayer at high MOI prior to RNA isolation, RT-PCR amplification of the region encompassing ORF4 and sequence analysis. The positions of the introduced stop codons in the mutants M1, M10 and M20 are boxed as are the sequences after 5 repeated passages in cell culture. (F) Western blot analysis of VF1 and VP2 expression in cells infected with either wild-type MNV or passage 5 VF1 mutant viruses M1, M10 and M20. 18 hours post infection at a high MOI (4 TCID₅₀ per cell) cells were harvested, separated by SDS-PAGE prior to western blot analysis using antisera to either VF1 or VP2. Note that batch-to-batch variation in the quality of the anti-VP2 antisera accounts for the variations in the levels of non-specific proteins detected in panels C and F.

(A) Confocal microscopy of cells transfected with either N- or C-terminal fusions of EGFP to VF1. Cos7 cells were transfected with plasmids expressing VF1-EGFP fusion proteins, prior to co-staining with Mitotracker, fixation and staining with DAPI. Cells were analyzed using a Zeiss Meta510 confocal microscope. GFP transfected and Mitotracker stained Cos7 cells are also shown as a control. (B) Biochemical fractionation demonstrates that VF1 localizes to mitochondria during virus infection. RAW264.7 cells were either mock infected or infected with wild-type MNV followed by mitochondrial purification 12 hours post infection. Samples were subsequently analyzed by western blot using antisera to VF1, apoptosis inducing factor 1 (AIF) or poly rC-binding protein1/2 (PCBP). Total cellular lysate was prepared by lysing cells directly in SDS-PAGE sample buffer and run alongside as a control.

(A) Time course of ISG54 and CXCL10 mRNA expression in RAW264.7 cells infected at an MOI of 0.1 TCID₅₀ per cell. mRNA levels were quantified by qPCR using an endogenous control gene (Hypoxanthine-guanine phosphoribosyltransferase, HPRT). Expression of the respective mRNAs was then calculated using the ΔΔCt method to compare infected and mock infected cells. Relative fold change was calculated using mock infected samples taken at comparable time points. Normalization was performed following MNV quantification in each sample. Infections were carried out in triplicate with each sample being subsequently analyzed in duplicate by qPCR. The error bars denote standard deviation from the mean. Statistical analysis was performed using an unpaired two tailed t test. (B) Time course of IFN-Beta mRNA and protein production in RAW264.7 cells infected as in (A). mRNA upregulation was calculated as described for (A). Interferon Beta protein was quantified by murine IFN-B specific ELISA at 24 hpi from supernatant samples taken from infected cells. For the ELISA infections were carried out in sextuplate. The error bars denote standard deviation from the mean. (C) MEF cells transfected with an IFN-Beta promoter driven luciferase reporter as well as expression constructs for RIG1, MDA5, MAVS or TBK1 were co-transfected with plasmid DNA expressing VF1 or blank message. Luciferase production was assayed 24 hours post transfection as described in the Materials and Methods. Transfections were carried out in triplicate. The error bars denote standard deviation from the mean. Statistical analysis was performed using an unpaired two tailed t test.

(A) Time course of caspase 3/7 activity in RAW264.7 cells treated with Staurosporine or infected with wild-type MNV-1 (WT) or a VF1 knockout virus (M1). Cells were infected at a MOI of 5 TCID₅₀ per cell and samples harvested at various times post infection. Caspase 3/7 activity was determined using a commercial assay from Promega (Glo 3/7) and the activity represented as relative light units (RLU). Each assay was performed in triplicate with the average and standard error within a single experiment plotted. Statistical analysis was performed using 2 way ANOVA with Bonfferoni post tests (B) Western blot analysis of extracts prepared from cells infected as detailed in (A). Samples were prepared at various times post infection, separated by SDS-PAGE and subsequently immune blotted using antisera to GAPDH, NS7, VP1 or VF1. Non-infected (Mock) cells were included as controls. (C). Caspase 3/7 activity assay in RAW 264.7 cells infected with M1 or WT MNV-1 or UV-inactivated M1 and WT viruses. Samples were analyzed at 16 hours post infection with triplicate samples taken. The mean and standard error within a single experiment are plotted. Statistical analysis was performed using an unpaired two tailed t-test (WT versus M1). P<0.01 is illustrated as ** and P<0.001 is shown as ***. Western blot analysis of GAPDH, NS7 and cleaved caspase 3 for the same samples is also shown.

Age and sex matched C57BL/6 mice were inoculated by oral gavage with 1×10⁷ TCID₅₀ of low passage, sequence verified, wild-type (WT) or VF1 knockout (M1) viruses. Body weight was measured on a daily basis and expressed as a percentage of the weight on day 0 prior to inoculation. The mean weight and the standard error are plotted as a line graph covering the duration of the experiment (A). Quantitative real-time RT-PCR quantification of viral genome copies in the mesenteric lymph node isolated from mice at various times post inoculation (B). The mean and standard error are plotted. Statistical analysis was performed using two-way ANOVA and Bonferroni post tests (WT versus M1). The blue line represents the detection limit as determined by the sensitivity of qPCR to detect standards, equating to 80 copies per µg of RNA. Samples that were below the detection limit were set as 80 copies per µg of RNA.

Age and sex matched STAT1-/- mice were inoculated by oral gavage with 1000 TCID₅₀ of low passage, sequence verified, wild-type (WT-v) or VF1 knockout (M1-v) viruses generated using a virulent backbone cDNA construct. As a measure of the severity of clinical disease, body weight was measured on a daily basis and expressed as percentage of the weight on day 0 prior to inoculation. Mock infected animals were orally inoculated with a control lysate preparation, generated as described in the materials and methods. For clarity the mean weight and the standard error are plotted as both a line graph covering the duration of the experiment (A) and as individual animal weights during days 3-6 (B). Statistical analysis was performed using two-way ANOVA with Bonferroni post-tests (WT-v versus M1-v). The hash and asterisk highlight animal number 776 and 751 used in the sequence analysis on days 5 and 7 respectively as described in the text. Note that animal 751 was removed from the study on day 7 (not shown on the plot) due to humane endpoints.

Quantitative real-time RT-PCR quantification of viral genome copies in various tissues isolated at 3 (A) or 5 (B) days post infections with either wild type (WT-v) or VF1 knockout (M1-v). At various times post inoculation, tissue samples were isolated and RNA extracted. The genome equivalent per µg of RNA was then determined by comparison to a standard curve generated from in vitro transcribed RNA. The blue line indicates the limit of the detection, based on the ability to detect viral RNA specifically in RNA samples prepared from mock infected animals. Each RNA sample was analyzed at least in duplicate and the mean taken. The mean of each group and standard error is plotted. MLN refers to the mesenteric lymph nodes.

Age and sex matched STAT1-/- mice were inoculated by oral gavage with 1000 TCID₅₀ of low passage, sequence verified, wild-type (WT-v) or VF1 knockout (M1-v) viruses generated using a virulent backbone cDNA construct. Necropsies were performed 5 days post infection. Spleen, small intestine and liver tissues were fixed in Bouin's solution, embedded in paraffin wax and sections stained with haematoxylin and eosin.