title: Knockdown of USP18 increases α 2a interferon signaling and induction of interferon-stimulating genes but does not increase antiviral activity in Huh7 cells.
abstract: The current standard of care for hepatitis C virus (HCV) patients is cotreatment with human alpha interferon (IFN-α) and ribavirin. The host factor USP18 functions to regulate the interferon signaling pathway by acting as an off-switch. In order to understand whether the inhibition of USP18 represents a valid target for the enhancement of interferon treatment for chronic viral diseases, we have used a wide range of RNA interference (RNAi) reagents to suppress USP18 gene expression in Huh7 cell lines. We demonstrate that a USP18 knockdown results in IFN-α2a signaling (measured by increased IFN-stimulated response element [ISRE] reporter gene activity, 2',5'-oligoadenylate synthetase [2-5 OAS] expression, and ISG15 induction) that is increased by ∼100-fold, whereas the antiviral (AV) potency in both the Huh7 HCV subgenomic replicon assay and the Huh7.5 HCV infectious virus assay increased by ∼3-fold. While the degree of the USP18 knockdown of USP18 elicited by the different RNAi reagents correlated with the enhancement of IFN-α2a signaling, it did not correlate with the enhancement of AV activity. The failure of increased IFN-α2a signaling to fully translate into increased AV potency was also observed for encephalomyocarditis virus (EMCV) assays using Huh7.5 cells. These data suggest that the IFN-mediated AV response in Huh7.5 cells has only a limited dependence on USP18 activity.
authors.affiliation: Internal Medicine, IPC 424, Pfizer PGRD, Discovery Biology, Sandwich Laboratories, Sandwich, Kent CT13 9NJ, United Kingdom. ted.murray@pfizer.com
authors.forename: E J
authors.initials: EJ
authors.lastname: Murray
authors.forename: Frances
authors.initials: F
authors.lastname: Burden
authors.forename: Nigel
authors.initials: N
authors.lastname: Horscroft
authors.forename: Caroline
authors.initials: C
authors.lastname: Smith-Burchnell
authors.forename: Mike
authors.initials: M
authors.lastname: Westby
mesh_terms: D000998:Antiviral Agents / Q000627:therapeutic use*; D015153:Blotting, Western; D002460:Cell Line; D010450:Endopeptidases / Q000235:genetics*; D006801:Humans; D016898:Interferon-alpha / Q000627:therapeutic use*; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012254:Ribavirin / Q000627:therapeutic use*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D043222:Ubiquitin Thiolesterase
chemical_list: D000998:Antiviral Agents; D016898:Interferon-alpha; D012254:Ribavirin; D010450:Endopeptidases; C485560:USP18 protein, human; D043222:Ubiquitin Thiolesterase