Abstract
Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.
Highlights
RNA viruses are considered to be one of the most severe threats to the human population and quality of life [1]
The proteolytic activity of the tick-borne encephalitis virus (TBEV) NS3 protease is dependent on association with the NS2B cofactor
(residues 45–96 linked with residues 116 to 131), followed by residues 1–190 of NS3 linked via a glycine-rich linker (Figure S1A). This construct is similar to the constructs previously used to express other active and soluble flaviviral proteases, such as dengue viruses (DENV) [29]
Summary
RNA viruses are considered to be one of the most severe threats to the human population and quality of life [1]. Since the beginning of this millennium, we have witnessed at least 60 epidemic outbreaks around the world, mostly caused by RNA viruses. Excluding the ongoing COVID-19 pandemic, these have caused more than a million deaths. The viruses responsible include: influenza virus, Ebola virus (EBOV), Zika virus (ZIKV), yellow fever virus (YFV), dengue viruses (DENV), measles, and coronaviruses such as SARS, MERS, and SARS-CoV-2. The effect on human health is devastating, just as is the economic burden of these epidemics. The global cost of COVID-19 alone is astronomical and is predicted to surpass the GDPs of Germany, the UK, and France combined. The global importance of targeting the RNA viruses is indisputable
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