A SARS-CoV-2 mini-genome assay based on negative-sense RNA to study replication inhibitors and emerging mutations

Vial, Thomas; Ms, Oade; Ca, Russell; Eggink, Dirk; Velthuis, Aartjan J.W. te

doi:10.1101/2021.06.28.450211

Cited by 3 publications

(1 citation statement)

References 54 publications

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“…Vial et al showed that the SARS-CoV-2 proteins required for the synthesis of positive strands from negative-strand replicon RNA are Nsp7, 8, and 12. Using these systems, they confirmed the anti-SARS-CoV-2 activity of remdesivir [ 36 ]. From these studies, it was concluded that the 5′-UTR, 3′-UTR, and reporter genes such as GFP and luciferase are required to produce SARS-CoV-2 replicon RNA, and that RNA-dependent RNA polymerases (RdRp, nsp12) are required for replication.…”

Section: Discussionmentioning

confidence: 83%

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

Furutani

Toguchi

Higuchi

et al. 2021

IJMS

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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.

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Section: Discussionmentioning

confidence: 83%

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

Furutani

Toguchi

Higuchi

et al. 2021

IJMS

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BPR3P0128, a non-nucleoside RNA-dependent RNA polymerase inhibitor, inhibits SARS-CoV-2 variants of concern and exerts synergistic antiviral activity in combination with remdesivir

Tang,

Chang,

Lin

et al. 2024

Antimicrob Agents Chemother

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Viral RNA-dependent RNA polymerase (RdRp), a highly conserved molecule in RNA viruses, has recently emerged as a promising drug target for broad-acting inhibitors. Through a Vero E6-based anti-cytopathic effect assay, we found that BPR3P0128, which incorporates a quinoline core similar to hydroxychloroquine, outperformed the adenosine analog remdesivir in inhibiting RdRp activity (EC 50 = 0.66 µM and 3 µM, respectively). BPR3P0128 demonstrated broad-spectrum activity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern. When introduced after viral adsorption, BPR3P0128 significantly decreased SARS-CoV-2 replication; however, it did not affect the early entry stage, as evidenced by a time-of-drug-addition assay. This suggests that BPR3P0128’s primary action takes place during viral replication. We also found that BPR3P0128 effectively reduced the expression of proinflammatory cytokines in human lung epithelial Calu-3 cells infected with SARS-CoV-2. Molecular docking analysis showed that BPR3P0128 targets the RdRp channel, inhibiting substrate entry, which implies it operates differently—but complementary—with remdesivir. Utilizing an optimized cell-based minigenome RdRp reporter assay, we confirmed that BPR3P0128 exhibited potent inhibitory activity. However, an enzyme-based RdRp assay employing purified recombinant nsp12/nsp7/nsp8 failed to corroborate this inhibitory activity. This suggests that BPR3P0128 may inhibit activity by targeting host-related RdRp-associated factors. Moreover, we discovered that a combination of BPR3P0128 and remdesivir had a synergistic effect—a result likely due to both drugs interacting with separate domains of the RdRp. This novel synergy between the two drugs reinforces the potential clinical value of the BPR3P0128-remdesivir combination in combating various SARS-CoV-2 variants of concern.

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Enisamium Inhibits SARS-CoV-2 RNA Synthesis

et al. 2021

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Pandemic SARS-CoV-2 causes a mild to severe respiratory disease called coronavirus disease 2019 (COVID-19). While control of the SARS-CoV-2 spread partly depends on vaccine-induced or naturally acquired protective herd immunity, antiviral strategies are still needed to manage COVID-19. Enisamium is an inhibitor of influenza A and B viruses in cell culture and clinically approved in countries of the Commonwealth of Independent States. In vitro, enisamium acts through metabolite VR17-04 and inhibits the activity of the influenza A virus RNA polymerase. Here we show that enisamium can inhibit coronavirus infections in NHBE and Caco-2 cells, and the activity of the SARS-CoV-2 RNA polymerase in vitro. Docking and molecular dynamics simulations provide insight into the mechanism of action and indicate that enisamium metabolite VR17-04 prevents GTP and UTP incorporation. Overall, these results suggest that enisamium is an inhibitor of SARS-CoV-2 RNA synthesis in vitro.

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A SARS-CoV-2 mini-genome assay based on negative-sense RNA to study replication inhibitors and emerging mutations

Cited by 3 publications

References 54 publications

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

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

BPR3P0128, a non-nucleoside RNA-dependent RNA polymerase inhibitor, inhibits SARS-CoV-2 variants of concern and exerts synergistic antiviral activity in combination with remdesivir

Enisamium Inhibits SARS-CoV-2 RNA Synthesis

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