A nanoluciferase SARS-CoV-2 for rapid neutralization testing and screening of anti-infective drugs for COVID-19

Xie, Xuping; Muruato, Antonio E.; Zhang, Xianwen; Lokugamage, Kumari G.; Fontes-Garfias, Camila R.; Zou, Jing; Liu, Jianying; Ren, Ping; Balakrishnan, Mini; Cihlář, Tomáš; Tseng, Chien-Te K.; Makino, Shinji; Menachery, Vineet D.; Bilello, John P.; Shi, Pei–Yong

doi:10.1101/2020.06.22.165712

Cited by 73 publications

(122 citation statements)

References 79 publications

(25 reference statements)

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“…While remdesivir strongly inhibited virus replication in a dose-dependent manner with an IC 50 value of 0.43 µM, famotidine had no measurable effect ( Figure 4B). Our results are consistent with previously reported studies in which remdesivir exerted a greater antiviral effect in human lung A549 cells than in Vero E6 cells 26 .…”

Section: Famotidine Does Not Inhibit Sars-cov-2 Replication In Cultursupporting

confidence: 94%

The Effect of Famotidine on SARS-CoV-2 Proteases and Virus Replication

Loffredo¹,

Lucero²,

Chen

et al. 2020

Preprint

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The lack of coronavirus-specific antiviral drugs has instigated multiple drug repurposing studies to redirect previously approved medicines for the treatment of SARS-CoV-2, the coronavirus behind the ongoing COVID-19 pandemic. A recent, large-scale, retrospective clinical study showed that famotidine, when administered at a high dose to hospitalized COVID-19 patients, reduced the rates of intubation and mortality. A separate, patient-reported study associated famotidine use with improvements in mild to moderate symptoms such as cough and shortness of breath. While a prospective, multi-center clinical study is ongoing, two parallel in silico studies have proposed one of the two SARS-CoV-2 proteases, 3CLpro or PLpro, as potential molecular targets of famotidine activity; however, this remains to be experimentally validated. In this report, we systematically analyzed the effect of famotidine on viral proteases and virus replication. Leveraging a series of biophysical and enzymatic assays, we show that famotidine neither binds with nor inhibits the functions of 3CLpro and PLpro. Similarly, no direct antiviral activity of famotidine was observed at concentrations of up to 200 μM, when tested against SARS-CoV-2 in two different cell lines, including a human cell line originating from lungs, a primary target of COVID-19. These results rule out famotidine as a direct-acting inhibitor of SARS-CoV-2 replication and warrant further investigation of its molecular mechanism of action in the context of COVID-19.

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Section: Famotidine Does Not Inhibit Sars-cov-2 Replication In Cultursupporting

confidence: 94%

The Effect of Famotidine on SARS-CoV-2 Proteases and Virus Replication

Loffredo¹,

Lucero²,

Chen

et al. 2020

Preprint

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“…1d ). On the one hand, our data confirmed the poor incorporation of Sofosbuvir by SARS-CoV-2 16,31 . On the other hand, ddhCTP showed an interesting increase in termination activity at physiological CTP concentration, opening great perspectives for ddhCTP as an effective anti-coronavirus NA with a low cytotoxicity.…”

Section: Resultssupporting

confidence: 78%

Inhibition of SARS-CoV-2 polymerase by nucleotide analogs: a single molecule perspective

Seifert

Bera

Nies

et al. 2020

Preprint

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SummaryCoronavirus Disease 2019 (COVID-19) results from an infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the third coronavirus outbreak to plague humanity this century. Currently, the most efficacious therapeutic against SARS-CoV-2 infection is the Remdesivir (RDV), an adenine-like ribonucleotide analogue that is very efficiently incorporated by the SARS-CoV-2 replicase. Understanding why RDV is so well incorporated will facilitate development of even more effective therapeutics. Here, we have applied a high-throughput, single-molecule, magnetic-tweezers platform to study thousands of cycles of nucleotide addition by the SARS-CoV-2 replicase in the absence and presence of RDV, a Favipiravir-related analog (T-1106), and the endogenously produced ddhCTP. Our data are consistent with two parallel catalytic pathways of the replicase: a high-fidelity catalytic (HFC) state and a low-fidelity catalytic (LFC) state, the latter allowing the slow incorporation of both cognate and non-cognate nucleotides. ddhCTP accesses HFC, T-1106 accesses LFC as a non-cognate nucleotide, while RDV efficiently accesses both LFC pathway. In contrast to previous reports, we provide unequivocal evidence against RDV functioning as a chain terminator. We show that RDV incorporation transiently stalls the replicase, only appearing as termination events when traditional, gel-based assays are used. The efficiency of ddhCTP utilization by the SARS-CoV-2 replicase suggests suppression of its synthesis during infection, inspiring new therapeutic strategies. Use of this experimental paradigm will be essential to the development of therapeutic nucleotide analogs targeting polymerases.

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“…We tested the suitability of the SARS-CoV-2 optimized reporter cell line to assess the antiviral activity of Remdesivir and determined an IC 50 of 16 nM in our reporter system. By using N staining as an alternative read-out, we obtained a somewhat lower efficacy of Remdesivir (IC 50 = 40 nM), which is closer to the data reported in the literature (42,43,48). The reduced sensitivity of the reporter construct, which relies on 3CL pro activity and GFP translocation, might stem from failure to detect cells with low levels of infection whereas the highly expressed N protein is already detectable by IF.…”

Section: Live Cell Imaging Of Sars-cov-2 Infectionsupporting

confidence: 85%

A versatile reporter system to monitor virus infected cells and its application to dengue virus and SARS-CoV-2

Pahmeier

Neufeldt

Cerikan

et al. 2020

Preprint

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Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of that are flaviviruses, such as dengue virus and Zika virus that cause millions of yearly infections and spread around the globe, and coronaviruses, such as SARS-CoV-2, which is the cause of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of virology research in determining mechanisms to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective interventions. Here, we describe the generation and characterization of a reporter system to visualize dengue virus and SARS-CoV-2 replication in live cells. The system is based on viral protease activity causing cleavage and nuclear translocation of an engineered fluorescent protein that is expressed in the infected cells. We show the suitability of the system for live cell imaging and visualization of single infected cells as well as for screening and testing of antiviral compounds. Given the modular building blocks, the system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility.IMPORTANCEReporter systems are useful tools for fast and quantitative visualization of viral replication and spread within a host cell population. Here we describe a reporter system that takes advantage of virus-encoded proteases that are expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the fluorescent protein translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.

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A nanoluciferase SARS-CoV-2 for rapid neutralization testing and screening of anti-infective drugs for COVID-19

Cited by 73 publications

References 79 publications

The Effect of Famotidine on SARS-CoV-2 Proteases and Virus Replication

The Effect of Famotidine on SARS-CoV-2 Proteases and Virus Replication

Inhibition of SARS-CoV-2 polymerase by nucleotide analogs: a single molecule perspective

A versatile reporter system to monitor virus infected cells and its application to dengue virus and SARS-CoV-2

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