Establishment of a Rapid Detection System for ISG20-Dependent SARS-CoV-2 Subreplicon RNA Degradation Induced by Interferon-α

• Published in: International journal of molecular sciences Vol. 22; no. 21; p. 11641
• Main Authors: Furutani, Yutaka, Toguchi, Mariko, Higuchi, Shoko, Yanaka, Kaori, Gailhouste, Luc, Qin, Xian-Yang, Masaki, Takahiro, Ochi, Sae, Matsuura, Tomokazu
• Format: Journal Article
• Language: English
• Published: Basel MDPI AG 01-11-2021; MDPI
• Subjects: CDM-3008; Coronaviruses; COVID-19; Cytomegalovirus; Degradation; Exonuclease; Gene expression; High-throughput screening; Interferon; interferon-stimulated genes; ISG20; Kinases; Nucleocapsids; Nucleotide sequence; replicon; Severe acute respiratory syndrome coronavirus 2; siRNA; Viral infections; α-Interferon; β-Interferon
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms222111641

Inhaled nebulized interferon (IFN)-α and IFN-β have been shown to be effective in the management of coronavirus disease 2019 (COVID-19). We aimed to construct a virus-free rapid detection system for high-throughput screening of IFN-like compounds that induce viral RNA degradation and suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We prepared a SARS-CoV-2 subreplicon RNA expression vector which contained the SARS-CoV-2 5′-UTR, the partial sequence of ORF1a, luciferase, nucleocapsid, ORF10, and 3′-UTR under the control of the cytomegalovirus promoter. The expression vector was transfected into Calu-3 cells and treated with IFN-α and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-α and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 μM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-α and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-α activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-α activity. Collectively, we constructed a virus-free rapid detection system to measure SARS-CoV-2 RNA suppression. Our data suggest that the SARS-CoV-2 subreplicon RNA was degraded by IFN-α-induced ISG20 exonuclease activity.