Direct Antiviral Activity of IFN-Stimulated Genes Is Responsible for Resistance to Paramyxoviruses in ISG15-Deficient Cells

Abstract

IFNs, produced during viral infections, induce the expression of hundreds of IFN-stimulated genes (ISGs). Some ISGs have specific antiviral activity, whereas others regulate the cellular response. Besides functioning as an antiviral effector, ISG15 is a negative regulator of IFN signaling, and inherited ISG15 deficiency leads to autoinflammatory IFNopathies, in which individuals exhibit elevated ISG expression in the absence of pathogenic infection. We have recapitulated these effects in cultured human A549-ISG15 2/2 cells and (using A549-UBA7 2/2 cells) confirmed that posttranslational modification by ISG15 (ISGylation) is not required for regulation of the type I IFN response. ISG15-deficient cells pretreated with IFN-a were resistant to paramyxovirus infection. We also showed that IFN-a 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 nonpermissive because of 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. Knockdown of IFIT1 restored parainfluenza virus 5 infection in IFN-a-pretreated, 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 pretreated with IFN-a for longer times, presumably because of inhibition of protein synthesis. These data show that the cause of virus resistance is 2-fold; ISG15 deficiency leads to the early overexpression of specific antiviral ISGs, but the later response is dominated by an unanticipated, ISG15-dependent loss of translational control.

Discovering antiviral restriction factors and pathways using genetic screens

“…Holthaus et al . confirmed that virus resistance in ISG15 −/− cells was due to the overexpression of viral restriction factors [114]. Because cells are resistant to infection, they are phenotypically silent and not detectable by FACS when infected with a reporter virus; deletion of a restriction factor, even one with low to moderate activity, results in a detectable signal that would be otherwise missed.…”

“…One possibility might be to use cells deficient in IFN stimulated gene 15 (ISG15); these cells, when treated with IFN-α, exhibit an overamplified IFN response resulting in the overexpression of ISGs and, subsequently, a greater ability to resist virus infection. Holthaus et al confirmed that virus resistance in ISG15 −/− cells was due to the overexpression of viral restriction factors [114]. Because cells are resistant to infection, they are phenotypically silent and not detectable by FACS when infected with a reporter virus; deletion of a restriction factor, even one with low to moderate activity, results in a detectable signal that would be otherwise missed.…”

“…An exception to this is PIV5, where IFIT1 is known to be the primary restriction factor [139]. Recently, Holthaus et al developed an infection model using this information to determine that PIV5 virus resistance is a result of direct antiviral activity [114]. Therefore, a greater understanding of the antiviral response and the specific restriction factors involved, through high-throughput genetic screening, may allow investigation of the wider IFN response.…”

More than Meets the ISG15: Emerging Roles in the DNA Damage Response and Beyond

“…In protein synthesis ISG15 can inhibit translation of individual proteins through modification of RNA-binding partners [ 118 ], as well as by suppressing global or mRNA-specific translation via ISGylation of key translational regulators ( Figure 3 , Section 6) [ 36 , 119 , 120 ]. This activity is largely thought to be an antiviral mechanism whereby translation of newly synthesized viral proteins is restricted via translational suppression following IFN stimulation.…”

A co-opted ISG15-USP18 binding mechanism normally reserved for deISGylation controls type I IFN signalling

“…Several reports now show that inherited ISG15-deficiency in humans causes type I interferonopathy and autoinflammatory disease (17)(18)(19). ISG15-deficient cells exhibited enhanced and prolonged ISG expression and a concomitant resistance to virus infection (18,20,21) a phenotype also associated with USP18 deficiency (13,22). Indeed, despite high levels of USP18 transcription, USP18 protein levels in ISG15-deficient cells are very low (18,20,21) and previous reports have shown that intracellular ISG15 is required for rescuing USP18 from S-phase kinase associated protein 2 (SKP2)-mediated proteasomal degradation (18,23).…”