Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4
Figure 5
VF1 antagonizes the innate immune response to MNV infection.
(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.