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Deprecated: Implicit conversion from float 265.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Int+J+Mol+Sci 2021 ; 22 (21): ä Nephropedia Template TP
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Establishment of a Rapid Detection System for ISG20-Dependent SARS-CoV-2 Subreplicon RNA Degradation Induced by Interferon-alpha #MMPMID34769072
Furutani Y; Toguchi M; Higuchi S; Yanaka K; Gailhouste L; Qin XY; Masaki T; Ochi S; Matsuura T
Int J Mol Sci 2021[Oct]; 22 (21): ä PMID34769072show ga
Inhaled nebulized interferon (IFN)-alpha and IFN-beta 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-alpha and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-alpha and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 muM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-alpha and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-alpha activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-alpha 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-alpha-induced ISG20 exonuclease activity.