AbstractInterferons (IFNs), produced during viral infections, induce the expression of hundreds of IFN- stimulated genes (ISGs). Some ISGs have specific antiviral activity while others regulate the cellular response. Besides functioning as an antiviral effector, IFN-stimulated gene 15 (ISG15) is a negative regulator of IFN signalling and inherited ISG15-deficiency leads to autoinflammatory interferonopathies where individuals exhibit elevated ISG expression in the absence of pathogenic infection. We have recapitulated these effects in cultured human A549-ISG15-/-cells and (using A549-UBA7-/-cells) confirmed that posttranslational modification by ISG15 (ISGylation) is not required for regulation of the type-I IFN response. ISG15-deficient cells pre-treated with IFN-α were resistant to paramyxovirus infection. We also showed that IFN-α treatment of ISG15-deficient cells led to significant inhibition of global protein synthesis leading us to ask whether resistance was due to the direct antiviral activity of ISGs or whether cells were non-permissive due to translation defects. We took advantage of the knowledge that IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) is the principal antiviral ISG for parainfluenza virus 5 (PIV5). Knockdown of IFIT1 restored PIV5 infection in IFN-α-pre-treated ISG15-deficient cells, confirming that resistance was due to the direct antiviral activity of the IFN response. However, resistance could be induced if cells were pre-treated with IFN-α for longer times, presumably due to inhibition of protein synthesis. These data show that the cause of virus resistance is two-fold; ISG15-deficiency leads to the ‘early’ over-expression of specific antiviral ISGs, but the later response is dominated by an unanticipated, ISG15- dependent, loss of translational control.Key pointsCell culture model of ISG15-deficiency replicate findings in ISG15-/-patient cellsCause of resistance in ISG15-/-cells differs depending on duration of IFN treatmentISG15-/-patients without serious viral disease don’t prove ISGylation is unimportant