ABSTRACT
Elucidating the molecular interactions between virus and host is fundamental to understanding the mechanism of viral pathogenesis. Here, we present a protocol to screen SARS-CoV-2 protein interactors using an antibody-based TurboID proximity labeling approach. This technique directly identifies biotinylated peptides labeled by the TurboID-tagged viral proteins. We describe the steps to prepare biotinylated peptide samples for mass spectrometry analysis and a stringent workflow to identify biotinylated high-confidence interactors of the virus by filtering out non-specific co-purified proteins. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).
Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies , COVID-19/diagnosis , Humans , Mass Spectrometry , Viral ProteinsABSTRACT
The SARS-CoV-2 coronavirus infects human cells through the interaction of the viral envelope spike protein (IPR044366) with the human angiotensin-converting enzyme 2 (ACE2), expressed at the surface of target cells. Here, we describe a detailed protocol to measure the binding of the receptor binding domain (RBD) of spike to ACE2 by time-resolved fluorescence resonance energy transfer (TR-FRET). The assay detects the spike/ACE2 interaction in physiologically relevant cellular contexts and is suitable for high-throughput investigation of interfering small-molecule compounds and antibodies. For complete details on the use and execution of this protocol, please refer to Cecon et al. (2021).
Subject(s)
Fluorescence Resonance Energy Transfer/methods , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/immunology , COVID-19/metabolism , HEK293 Cells , Humans , Protein Binding/physiology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/immunologyABSTRACT
This protocol describes an in vitro fluorogenic assay to measure the proteolytic activity and identify inhibitors of Mpro, the main protease produced by SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2). Studies to identify potential inhibitors of Mpro mainly rely on in silico molecular dynamics simulations or on FRET (Fluorescence Resonance Energy Transfer) substrates. The protocol is based on an aminomethyl coumarin substrate. High sensitivity, specificity, and an easily detectable fluorescent read-out are the advantages offered by this rapid assay, which allows high throughput screening of new Mpro inhibitors.
Subject(s)
Antiviral Agents/pharmacology , COVID-19 Drug Treatment , Coronavirus 3C Proteases/antagonists & inhibitors , Fluorescence Resonance Energy Transfer/methods , Protease Inhibitors/pharmacology , SARS-CoV-2/enzymology , Viral Proteins/antagonists & inhibitors , COVID-19/metabolism , COVID-19/virology , Coronavirus 3C Proteases/metabolism , Coumarins/metabolism , Drug Discovery , High-Throughput Screening Assays , Humans , In Vitro TechniquesABSTRACT
We present a protocol for analyzing the impact of SARS-CoV-2 proteins in interferon signaling using luciferase reporter assays. Here, the induction of defined promoters can be quantitatively assessed with high sensitivity and broad linear range. The results are similar to those obtained using qPCR to measure endogenous mRNA induction. The assay requires stringent normalization and confirmation of the results in more physiological settings. The protocol is adaptable for other viruses and other innate immune stimuli. For complete details on the use and execution of this protocol, please refer to Hayn et al. (2021).