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Biochem J ; 479(8): 901-920, 2022 04 29.
Article in English | MEDLINE | ID: covidwho-1774010


Diagnostic testing continues to be an integral component of the strategy to contain the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) global pandemic, the causative agent of Coronavirus Disease 2019 (COVID-19). The SARS-CoV-2 genome encodes the 3C-like protease (3CLpro) which is essential for coronavirus replication. This study adapts an in vitro colorimetric gold nanoparticle (AuNP) based protease assay to specifically detect the activity of SARS-CoV-2 3CLpro as a purified recombinant protein and as a cellular protein exogenously expressed in HEK293T human cells. We also demonstrate that the specific sensitivity of the assay for SARS-CoV-2 3CLpro can be improved by use of an optimised peptide substrate and through hybrid dimerisation with inactive 3CLpro mutant monomers. These findings highlight the potential for further development of the AuNP protease assay to detect SARS-CoV-2 3CLpro activity as a novel, accessible and cost-effective diagnostic test for SARS-CoV-2 infection at the point-of-care. Importantly, this versatile assay could also be easily adapted to detect specific protease activity associated with other viruses or diseases conditions.

COVID-19 , Metal Nanoparticles , Antiviral Agents , COVID-19/diagnosis , Colorimetry , Coronavirus 3C Proteases , Gold , HEK293 Cells , Humans , Peptide Hydrolases , Protease Inhibitors , SARS-CoV-2
PLoS Pathog ; 18(2): e1010265, 2022 02.
Article in English | MEDLINE | ID: covidwho-1686115


Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.

Biosensing Techniques/methods , COVID-19 Testing/methods , COVID-19/virology , Luminescent Measurements/methods , Peptide Hydrolases/analysis , SARS-CoV-2/enzymology , Viral Proteins/analysis , COVID-19/diagnosis , Cell Line , Humans , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Replication