Vasou, A. | bioRxiv Science

bioRxiv2024

Highly-sensitive reporter cell line for detection of interferon types I-III and their neutralization by antibodies

Interferons (IFNs) are a critical component of innate immune defenses and limit viral disease severity. To advance studies on IFNs and their neutralization by pathogenic autoantibodies, we generated a Renilla luciferase-based reporter cell line capable of detecting the activities of IFN-Is, IFN-II, and IFN-IIIs. The reporter cell line exhibits a 125-to 2000-fold higher sensitivity to IFNs than a commonly-used alternative biological reporter system, and allows for a rapid and simple live-cell workflow for detecting low titer amounts of neutralizing anti-IFN antibodies.

immunology↗

bioRxiv2021

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

Type I interferon (IFN) signalling induces the expression of several hundred IFN-stimulated genes (ISGs) that provide an unfavourable environment for viral replication. To prevent an overexuberant response and autoinflammatory disease, IFN signalling requires tight control. One critical regulator is the ubiquitin-like protein ISG15, evidenced by autoinflammatory disease in patients with inherited ISG15 deficiencies. Current models suggest that ISG15 stabilises USP18, a well-established negative regulator of IFN signalling. USP18 also functions as an ISG15-specific peptidase that cleaves ISG15 from ISGylated proteins; however, USP18s catalytic activity is dispensable for controlling IFN signalling. Here, we show that the ISG15-dependent stabilisation of USP18 involves transient hydrophobic interactions. Nonetheless, while USP18 stabilisation is necessary, it is not sufficient for regulation of IFN signalling. USP18 requires non-covalent interactions with the ISG15 C-terminal diGlycine motif to promote its regulatory function. This trait may have been acquired in humans through co-option of a binding mechanism normally reserved for deISGylation, identifying an unexpected new function for human ISG15.

immunology↗

bioRxiv2019

Direct antiviral activity of interferon stimulated genes is responsible for resistance to paramyxoviruses in ISG15-deficient cells

Interferons (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- were resistant to paramyxovirus infection. We also showed that IFN- 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--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- 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 cells Cause of resistance in ISG15-/- cells differs depending on duration of IFN treatment ISG15-/- patients without serious viral disease dont prove ISGylation is unimportant

immunology↗