How fluorescent proteins can be applied in SARS-CoV-2 research

GFP or RFP and their derivatives are commonly used for virus research

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Fluorescent proteins (FPs) like GFP or RFP and their derivatives are commonly used for virus research. Frequently, they are applied as fluorescent markers in microscopy or cell sorting experiments and as protein tags for protein purification, immunoprecipitation or protein-protein interaction assays. Here, we provide a short outline that describes how FPs have been used in the current SARS-CoV-2 research. Please note that this summary does not provide a complete overview; also, many referenced papers are pre-prints without peer reviews.

Application examples of fluorescent proteins in SARS-CoV-2 research

Virus protein purification

Although fluorescent protein tags are rarely applied in protein purification, the yellow fluorescent protein (YFP) has been used for purification of the receptor-binding domain (RBD) of the spike glycoprotein S [DOI: 10.1101/2020.04.16.045419]. Here, RBD-vYFP was expressed in mammalian cells and isolated from cell extract with an anti-GFP Nanobody resin (such as GFP-Trap). The RBD-vYFP fusion protein was eluted from the resin with an acidic buffer. Efforts to cleave off YFP failed and the full-length RBD-vYFP fusion was used for additional assays.

ChromoTek’s  Nano-Traps  such as  GFP-Trap  is well suited for the purification of FP-tagged virus proteins like RBD. Besides protein purification, the Nano-Traps can be used for immunoprecipitation of FP-tagged virus proteins and to find potential viral and host cell binding partners.

Fluorescence microscopy

FPs are ideal tools for visualization of virus proteins in microscopy and used as reporters. Two approaches are reported:

• The virus is labeled with the FP by introducing the FP reporter gene into the viral genome. When the virus infects cells and viral proteins are produced, also the FP is expressed. The resulting fluorescent signal labels the infected cells.
• Viral proteins are tagged with the FP and recombinantly expressed in cells. The FP signal can be used to analyze the location of the virus protein, for characterization and to find binding partners.

The downsides of FPs are their photobleaching and relative weak signal intensity in super-resolution microscopy. ChromoTek’s  Nano-Boosters, e.g.  GFP-Booster, which is a GFP Nanobody conjugated to fluorescent dyes, overcome these issues by locating a fluorescent dye to the FPs, which stabilizes and increases signal intensity.

Flow cytometry/ FACS

FP tags can also be used in flow cytometry/ FACS because cells are detected based on the fluorescent signal resulting from intracellular or cell surface FPs:

• Cells are infected by the virus coding for FP tagged viral protein(s). Cells, which express the FP, are counted or sorted based on the fluorescent signal.
• Cells expressing FP tagged virus proteins can be recorded or sorted.
• Cells are incubated with FP tagged viral proteins. Cells, which have a fluorescent signal based from the FP tagged viral protein binding to molecules on the cell surface are counted.

If the fluorescent signal of the FP is not strong enough or another fluorescent channel shall be used ChromoTek’s  Nano-Booster  can be applied. For example, if GFP shall be analyzed in the red channel, the GFP-Booster Alexa Fluor^® 647 can be used to locate a red fluorescent dye to the GFP fusion protein via the GFP Nanobody.

Overview of publications using FPs

Title	Publication Date	Fluorescent protein
Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies	05.05.2020	EGFP
PSGL-1 blocks SARS-CoV-2 S protein-mediated virus attachment and infection of target cells	02.05.2020	GFP (TurboGFP)
Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza	29.04.2020	GFP, mCherry
Inhibition of PIKfyve kinase prevents infection by EBOV and SARS-CoV-2	28.04.2020	eGFP, TagRFP
TMPRSS2 and TMPRSS4 mediate SARS-CoV-2 infection of human small intestinal enterocytes	23.04.2020	GFP
Synthetic nanobodies targeting the SARS-CoV-2 receptor-binding domain	18.04.2020	YFP
Human organs-on-chips as tools for repurposing approved drugs as potential influenza and COVID 19 therapeutics in viral pandemics	15.04.2020	GFP
Analysis of SARS-CoV-2-controlled autophagy reveals spermidine, MK-2206, and niclosamide as putative antiviral therapeutics	15.04.2020	EGFP, mRFP
An Infectious cDNA Clone of SARS-CoV-2	13.04.2020	mNeonGreen
Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells	13.04.2020	EGFP
Potential host range of multiple SARS-like coronaviruses and an improved ACE2-Fc variant that is potent against both SARS-CoV-2 and SARS-CoV-1	11.04.2020	GFP
Vulnerabilities of the SARS-CoV-2 virus to proteotoxicity – opportunity for repurposed chemotherapy of COVID-19 infection	09.04.2020	EGFP
The sequence of human ACE2 is suboptimal for binding the S spike protein of SARS coronavirus 2	06.04.2020	sfGFP
An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice	03.04.2020	RFP, GFP
Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion	30.03.2020	EGFP
Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV	27.03.2020	EGFP
A human monoclonal antibody blocking SARS-CoV-2 infection	12.03.2020	GFP
LY6E impairs coronavirus fusion and confers immune control of viral disease	07.03.2020	TagRFP, GFP, mCherry
Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B beta coronaviruses	24.02.2020	GFP
Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform	21.02.2020	GFP