Spike-mediated cellular infection and fusion among SARS-CoV-2 variants

Last updated: April 2022

Pseudoparticle infection and cell-fusion assays

InvivoGen offers a collection of biological tools to assess two key functions of the SARS-CoV-2 Spike protein: infection of target cells and cell fusion. Depending on your needs, you may choose among families of:

• SARS-CoV-2 permissive (target) cells

• Spike pseudotyping vectors

• SARS-CoV-2 "fusogenic" cells

Below are the data obtained with target cells exposed to Spike from the Wuhan original strain as well as from two variants of concern (VOCs), Delta and Omicron, which have been reported to differ in their infection and pathogenic capabilities.

SARS-CoV-2 permissive (target) cells:

InvivoGen has developed a series of target cells expressing high levels of the host receptor ACE2 alone, or in combination with the host protease TMPRSS2 at their cell surface. Thus, these cells are permissive to infection by SARS-CoV-2 and Spike-pseudotyped lentiviral particles (Figure 1). Moreover, they allow cell fusion and formation of syncytia (Figure 2), illustrating one major SARS-COV-2 cytopathic effect.

SARS-CoV-2 permissive cells express NF-κB- and IRF-inducible reporter genes encoding the Lucia™ luciferase or the secreted embryonic alkaline phosphatase (SEAP) to assess innate sensing pathways, or Spike-mediated fusogenicity using InvivoGen's cell fusion assays (Figure 3). These cells are also available without reporter genes.

HEK293-derived cell lines:

HEK-Blue™ hACE2(-TMPRSS2) cells express an NF-κB-inducible SEAP reporter gene.
HEK-Lucia™ hACE2(-TMPRSS2) cells express an NF-κB-inducible Lucia luciferase reporter gene. Available upon request.
HEK-hACE2(-TMPRSS2) cells do not express any inducible reporter gene. Available upon request.

A5493-derived cell lines:

A549-hACE2-(TMPRSS2) cells do not express any inducible reporter gene.
A549-Dual™ (KO-MDA5 or KO-RIG-I) hACE2-TMPRSS2 cells express two inducible reporter genes, allowing the concomitant study of the IRF and NF-κB pathways by monitoring the Lucia™ luciferase and SEAP, respectively.

Spike pseudotyping vectors for lentiviral assays:

InvivoGen offers a collection of pLV-Spike plasmids specifically designed for pseudotyping lentiviral particles with the full-length SARS-CoV-2 Spike protein from various variants. This family encompasses the first identified SARS-CoV-2 (Wuhan) and major variants that rapidly arose during the pandemic progression. Spike-mediated cell infection has been tested in-house using pLV-Spike plasmids for the Wuhan, Delta and Omicron variants together with ACE2(-TMPRSS2)-expressing HEK293-derived cells as permissive cells (Figure 1).

pLV-Spike (D614) encodes the Spike protein from the Wuhan (original) variant.
pLV-SpikeV8 encodes the Spike protein from the Delta (B.1.617.2) variant.
pLV-SpikeV11 encodes the Spike protein from the Omicron (BA.1) variant.

Browse the whole collection of pLV-Spike plasmids.

SARS-COV-2 Spike "fusogenic" cells:

InvivoGen has developed a simple cell fusion assay to assess SARS-CoV-2 Spike-mediated fusogenicity. Briefly, fusogenic ("donor") cells expressing Spike at the cell surface and a constitutively active human MyD88 adaptor molecule in the cytosol are co-cultured with permissive SARS-CoV-2 target ("acceptor") cells expressing an NF-kB-SEAP or -Lucia™ luciferase inducible reporter gene. Upon Spike-ACE2-dependent cell fusion, the active MyD88 adaptor is transferred from the "donor" to the "acceptor" cells allowing the reporter gene expression.

Depending on your needs, you may choose from "fusogenic" cells either transiently or stably transfected with a gene encoding the SARS-CoV-2 Spike protein:

- To compare the fusogenicity of SARS-CoV-2 variants, Spike-mediated cell fusion can be tested using MyD88-expressing HEK293 cells transiently transfected with Spike plasmids and HEK-Blue™ or -Lucia™ hACE2(-TMPRSS2) cells (Figure 3).

- To screen for antibodies or small molecules inhibiting cell fusion, MyD88-expressing HEK293 cells with stable Spike expression can be used as these have been selected on their fusogenic potency. "Donor" cells with stable expression of the Wuhan (original), Delta, or Omicron Spike have been functionally tested in a cell-fusion inhibition assay using the small molecule EK1C4 (Figure 4).

MyD88-expressing HEK293 cells:

293-hMyD88 cells express a constitutively active human MyD88 adaptor molecule.

Spike plasmids for cellular expression:

pUNO1-Spike encodes the Spike protein from the Wuhan (original) variant.
pUNO1-SpikeV8 encodes the Spike protein from the Delta (B.1.617.2) variant.
pUNO1-SpikeV11 encodes the Spike protein from the Omicron (BA.1) variant.
pUNO1-SpikeV12 encodes the Spike protein from the Omicron (BA.2) variant.

Browse the whole collection of pUNO1-Spike plasmids.

MyD88-expressing HEK293 cells with stable Spike expression:

Figure 1: Spike-mediated cellular infection

Infection of HEK cells expressing SARS-CoV-2 receptors by Spike-pseudotyped lentiviral particles. 2 x 10⁴ HEK-Blue™ hACE2 and HEK-Blue™ hACE2-TMPRSS2 cells (A), or HEK-hACE2 and HEK-hACE2-TMPRSS2 cells (B and C), were cultured in the presence of Wuhan (A), Delta (B), or Omicron (C) Spike-pseudotyped GFP lentiviral particles. After 72 hours the transduction efficiency of the Spike-pseudotyped GFP particles was evaluated by flow cytometry. Independent experiments are shown in each panel.

Figure 2: Spike-mediated cell fusion assessed by fluorescence microscopy

Spike-induced cellular fusion and syncitia formation in SARS-CoV-2 target cells. 2 x10⁴HEK-hACE2 cells (A) or HEK-hACE2-TMPRSS2 cells (B) were transiently transfected with a GFP encoding plasmid and incubated with 1 x10⁵293-hMyD88-S (Wuhan), 293-hMyD88-S-V8 (Delta), or 293-hMyD88-S-V11 (Omicron BA.1) cells at 37°C, 5% C0₂. After overnight incubation cellular fusion was evaluated by fluorescence microscopy. Syncytia are outlined with white broken lines.

Figure 3: Spike-mediated cell fusion reporter assay

Spike-induced cellular fusion in SARS-CoV-2 reporter target cells. 2 x 10⁴ HEK-Blue™ hACE2-TMPRSS2 cells (A), or HEK-Lucia™ hACE2-TMPRSS2 cells (B) were cultured with an increasing number of 293-hMyD88 "donor" cells transiently transfected with plasmids encoding SARS-CoV-2 Spike (Wuhan, original), SpikeV8 (Delta), SpikeV11 (Omicron BA.1), SpikeV12 (Omicron BA.2) or GFP. After overnight incubation, the cellular fusion was assessed by measuring the activity of SEAP and Lucia™ luciferase reporter activity in the supernatant using QUANTI-Blue™ Solution and QUANTI-Luc™ detection reagents, respectively. Data represent the fold increase of reporter signal over non-induced reporter target cells.

Figure 4: Dose-dependent inhibition of Spike-mediated cell fusion by EK1C4

EK1C4 inhibits Spike-induced cellular fusion in SARS-CoV-2 reporter target cells. 1 x 10⁴ HEK-Blue™ hACE2-TMPRSS2 cells (A), or HEK-Lucia™ hACE2-TMPRSS2 cells (B) were cultured with 2 x 10⁴ 293-hMyD88-S (Wuhan), 293-hMyD88-S-V8 (Delta), or 293-hMyD88-S-V11 (Omicron BA.1) "donor" cells in the presence of increasing concentrations of EK1C4. After overnight incubation, the cellular fusion was assessed by measuring the activity of SEAP and Lucia™ luciferase reporter activity in the supernatant using QUANTI-Blue™ Solution and QUANTI-Luc™ detection reagents, respectively. Data are shown as a percentage of maximal inhibition of cell-cell fusion.