Generation of SARS-CoV-2 reporter replicon for high-throughput antiviral screening and testing

He, Xi; Quan, Shuo; Xu, Min; Rodriguez, Silveria; Goh, Shih Lin; Wei, Jiajie; Fridman, Arthur; Koeplinger, Kenneth A.; Carroll, Steve; Grobler, Jay A.; Espeseth, Amy S.; Olsen, David B.; Hazuda, Daria J.; Wang, Dai

doi:10.1073/pnas.2025866118

Cited by 76 publications

(103 citation statements)

References 36 publications

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“…3B). This observation is in line with the remdesivir IC50 observed in virus infections and other replicon assays (25,26). To confirm that the reduction in Nluc activity level was correlated with a reduction in the positive-sense Nluc-N reporter RNA level, we performed an RT-qPCR analysis on the remdesivir-treated cells.…”

Section: Inhibition Of Mini-genome Assay By Antiviral Compoundssupporting

confidence: 79%

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

Vial

et al. 2021

Preprint

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Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus and the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Efforts to identify inhibitors of SARS-CoV-2 replication enzymes and better understand the mechanisms underlying viral RNA synthesis have largely relied on biosafety level 3 (BSL3) laboratories, limiting throughput and accessibility. Recently, replicon systems have been proposed that involve ~30 kb RNA-based replicons or large plasmids that express the viral structural and non-structural proteins (nsp) in addition to a positive-sense reporter RNA. Unfortunately, these assays are not user-friendly due to plasmid instability or a poor signal to background ratio. We here present a simple mini-genome assay consisting of a ~2.5 kb-long negative-sense, nanoluciferase-encoding sub-genomic reporter RNA that is expressed from a plasmid, and amplified and transcribed by the SARS-CoV-2 RNA polymerase core proteins nsp7, nsp8 and nsp12. We show that expression of nsp7, 8 and 12 is sufficient to obtain robust positive- and negative-sense RNA synthesis in cell culture, and that replication of the reporter RNA can be inhibited by active site mutations in nsp12 or the SARS-CoV-2 replication inhibitor remdesivir. The mini-genome assay provides a signal that is 170-fold above background on average, providing excellent sensitivity for high-throughput screens, while the use of small plasmids facilitates site-directed mutagenesis for fundamental analyses of SARS-CoV-2 RNA synthesis.

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Section: Inhibition Of Mini-genome Assay By Antiviral Compoundssupporting

confidence: 79%

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

Vial

et al. 2021

Preprint

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“…The major advantage of these approaches is speed and the number of potential candidate molecules identified, but these techniques need to be always coupled with relevant functional testing assays to triage the hits and validate their functional relevance. Newly developed cell-based replicon assays for SARS-CoV-2 should be valuable to characterize activity of candidate compounds and certify cell-based activity and activation of candidate prodrugs [ 27 , 28 ].…”

Section: Viral Replication Machinerymentioning

confidence: 99%

Report of the National Institutes of Health SARS-CoV-2 Antiviral Therapeutics Summit

Hall

Anderson

et al. 2021

The Journal of Infectious Diseases

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The NIH Virtual SARS-CoV-2 Antiviral Summit, held on November 6, 2020, was organized to provide an overview on the status and challenges in developing antiviral therapeutics for COVID-19, including combinations of antivirals. Scientific experts from the public and private sectors convened virtually during a live videocast to discuss SARS-CoV-2 targets for drug discovery as well as the preclinical tools needed to develop and evaluate effective small molecule antivirals. The goals of the Summit were to review the current state of the science, identify unmet research needs, share insights and lessons learned from treating other infectious diseases, identify opportunities for public-private partnerships, and assist the research community in designing and developing antiviral therapeutics. This report includes an overview of therapeutic approaches, individual panel summaries, and a summary of the discussions and perspectives on the challenges ahead for antiviral development.

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“…Recently, GC376, a 3CL protease inhibitor under commercial development for FIP, was reported to have anti–SARS-CoV-2 activity by us ( 6 ) and other groups ( 22 , 38 , 39 ), which suggests this compound is a lead compound for COVID-19 amenable to further optimization. The in vitro antiviral effects of GC376 are comparable to other remdesivir and other nucleoside analogs and protease inhibitors under development against SARS-CoV-2 ( 38 , 40 – 42 ). The antiviral compounds, including GC376, showed antiviral effects against SARS-CoV-2 in human ACE-2–expressing transgenic mice ( 27 , 40 , 43 ) or in rhesus macaques ( 25 ) with nonlethal infection.…”

Section: Discussionmentioning

confidence: 88%

“…The (38,(40)(41)(42). The antiviral compounds, including GC376, showed antiviral effects against SARS-CoV-2 in human ACE-2-expressing transgenic mice (27,40,43) or in rhesus macaques ( 25) with nonlethal infection.…”

Section: Discussionmentioning

confidence: 99%

Postinfection treatment with a protease inhibitor increases survival of mice with a fatal SARS-CoV-2 infection

Dampalla

Zheng

Perera

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a serious global public health threat. The 3C-like protease (3CLpro) is a virus protease encoded by SARS-CoV-2, which is essential for virus replication. We have previously reported a series of small-molecule 3CLpro inhibitors effective for inhibiting replication of human coronaviruses including SARS-CoV-2 in cell culture and in animal models. Here we generated a series of deuterated variants of a 3CLpro inhibitor, GC376, and evaluated the antiviral effect against SARS-CoV-2. The deuterated GC376 displayed potent inhibitory activity against SARS-CoV-2 in the enzyme- and the cell-based assays. The K18-hACE2 mice develop mild to lethal infection commensurate with SARS-CoV-2 challenge doses and were proposed as a model for efficacy testing of antiviral agents. We treated lethally infected mice with a deuterated derivative of GC376. Treatment of K18-hACE2 mice at 24 h postinfection with a derivative (compound 2) resulted in increased survival of mice compared to vehicle-treated mice. Lung virus titers were decreased, and histopathological changes were ameliorated in compound 2–treated mice compared to vehicle-treated mice. Structural investigation using high-resolution crystallography illuminated binding interactions of 3CLpro of SARS-CoV-2 and SARS-CoV with deuterated variants of GC376. Taken together, deuterated GC376 variants have excellent potential as antiviral agents against SARS-CoV-2.

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Generation of SARS-CoV-2 reporter replicon for high-throughput antiviral screening and testing

Cited by 76 publications

References 36 publications

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

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

Report of the National Institutes of Health SARS-CoV-2 Antiviral Therapeutics Summit

Postinfection treatment with a protease inhibitor increases survival of mice with a fatal SARS-CoV-2 infection

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