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

Posted by Dr. Astrid Sitte on May 14, 2020 12:28:52 PM
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.

SARS-CoV-2+GFP

 

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

Weblink

Structural Basis for Potent Neutralization of
Betacoronaviruses by Single-Domain Camelid
Antibodies

05.05.2020

EGFP

https://doi.org/10.1016/j.cell.2020.04.031

PSGL-1 blocks SARS-CoV-2 S protein-mediated virus attachment and
infection of target cells

02.05.2020

GFP (TurboGFP)

https://doi.org/10.1101/2020.05.01.073387

Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza

29.04.2020

GFP, mCherry

https://doi.org/10.1016/j.cell.2020.04.020

Inhibition of PIKfyve kinase prevents infection by EBOV and SARS-CoV-2

28.04.2020

eGFP, TagRFP

https://doi.org/10.1101/2020.04.21.053058

TMPRSS2 and TMPRSS4 mediate SARS-CoV-2 infection of human small intestinal enterocytes

23.04.2020

GFP

https://doi.org/10.1101/2020.04.21.054015

Synthetic nanobodies targeting the SARS-CoV-2 receptor-binding domain

18.04.2020

YFP

https://doi.org/10.1101/2020.04.16.045419

Human organs-on-chips as tools for repurposing approved drugs as potential influenza and COVID 19 therapeutics in viral pandemics

15.04.2020

GFP

https://doi.org/10.1101/2020.04.13.039917

Analysis of SARS-CoV-2-controlled autophagy reveals spermidine, MK-2206, and niclosamide as putative antiviral therapeutics

15.04.2020

EGFP, mRFP

https://doi.org/10.1101/2020.04.15.997254

An Infectious cDNA Clone of SARS-CoV-2

13.04.2020

mNeonGreen

https://doi.org/10.1016/j.chom.2020.04.004

Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

13.04.2020

EGFP

https://doi.org/10.1101/2020.04.08.026948

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

https://doi.org/10.1101/2020.04.10.032342

Vulnerabilities of the SARS-CoV-2 virus to proteotoxicity – opportunity for repurposed chemotherapy of COVID-19 infection

09.04.2020

EGFP

https://doi.org/10.1101/2020.04.07.029488

The sequence of human ACE2 is suboptimal for binding the S spike protein of SARS coronavirus 2

06.04.2020

sfGFP

https://doi.org/10.1101/2020.03.16.994236

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

https://doi.org/10.1126/scitranslmed.abb5883

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

https://doi.org/10.1038/s41422-020-0305-x

Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

27.03.2020

EGFP

https://doi.org/10.1038/s41467-020-15562-9

A human monoclonal antibody blocking SARS-CoV-2 infection

12.03.2020

GFP

https://doi.org/10.1101/2020.03.11.987958

LY6E impairs coronavirus fusion and confers immune control of viral disease

07.03.2020

TagRFP, GFP, mCherry

https://doi.org/10.1101/2020.03.05.979260

Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses

24.02.2020

GFP

https://doi.org/10.1038/s41564-020-0688-y

Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform

21.02.2020

GFP

https://doi.org/10.1101/2020.02.21.959817

 

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