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To better understand the mechanism of SARS-CoV-2 cell entry, BPS Bioscience has developed 3 new pseudotyped lentiviruses, allowing for the study of the interaction between  coronavirus Spike protein and human ACE2 protein in a physiologically relevant context. These lentiviruses include the ACE2 LentivirusSpike (SARS-CoV-2) Pseudotyped Lentivirus (Luciferase Reporter), and the Bald Lentiviral Pseudovirion (Luciferase Reporter).


ACE2 Lentivirus

ACE2 LentivirusLocalized on the surface of many human cell types (lungs, arteries, heart, kidney, and intestines), ACE2 is the entry point of the virus due to its interaction with the coronavirus Spike membrane protein. The ACE2 Lentivirus is replication incompetent, HIV-based, VSV-G pseudotyped lentiviral particles that are ready to be transduced into almost all types of mammalian cells, including primary and non-dividing cells. Under the control of EF1a promoter, the particles contain an ACE2 gene (NM_021804.3) allowing a transient expression of ACE2 in your target cell or a generation of a stable cell line expressing ACE2 with Puromycin.
Lentivirus Figure: Schematic of the lenti-vector used to generate the ACE2 lentivirus
 

ACE2 Lentivirus Data

The transduction efficiency and the expression of ACE2 in HEK293 cells has been evaluated by FACS analysis with two different strategies. The first used a specific anti-human ACE2 antibody (figure A). The transduced cells were stained by anti-human ACE2 polyclonal goat IgG primary antibody and Alexa Fluor 488-conjugated rabbit anti-goat IgG as a secondary antibody. In the second strategies (figure B), the transduced cells were stained by a specific ACE2 ligand (biotinylated Spike S1 protein) and the Phycoerythrin (PE) conjugated to Streptavidin for the detection. In both cases FACS analysis reveal a high level of ACE2 expression at the surface of the HEK293 cells.

Figures A & B: FACS analysis of the ACE2 expression in HEK293 cells transduced with ACE2 lentivirus. Blue: HEK293 parental cells; Green: HEK293 cells transduced with ACE2 lentivirus.

Spike (SARS-CoV-2) Pseudotyped Lentivirus (Luciferase Reporter)

SPIKE LentivirusDue to its interaction with the Human ACE2, the coronavirus Spike protein is involved in the first step of the viral replication, which is the attachment of the virus to the host cell. In combination with the ACE2 lentivirus, BPS Bioscience has developed the Spike (SARS-CoV-2) Pseudotyped Lentivirus. Here, the commonly used VSV-G viral fusion protein, which classically mediates the fusion between the virus envelope and host cellular membrane, has been replaced by the SARS-CoV-2 Spike protein (Genbank Accession #QHD43416.1) as the envelope glycoproteins. These pseudovirions also contain the firefly luciferase gene driven by a CMV promoter, to easily measure the spike-mediated cell entry via luciferase reporter activity. Luciferase detection was measured with the ONE-Step Luciferase reagentLentivirus Figure: Schematic of the Luciferase Reporter in SARS-CoV-2 Spike Pseudovirion.

Spike Lentivirus DataTransduction efficiency of the Spike (SARS-CoV-2) pseudotyped lentivirus has been evaluated on HEK293 cells expressing or not expressing the ACE2 protein (figure C) and on Calu3 cells (human lung cancer cell line which are epithelial and can act as respiratory models in preclinical applications) (figure D) used as a transduction control. Based on luciferase measurements, it appears that the Spike (SARS-CoV-2) pseudotyped lentivirus transduced ACE2-HEK293 and Calu3 cells with much greater efficiency compared with HEK293 parental cells. It indicates that the transduction is dependent upon ACE2 expression. The bald lentiviral pseudovirion, where no envelope glycoprotein is expressed, was used as a negative control.

Spike Lentivirus Data

With ACE2 dependent transduction efficiency proved, several neutralization assays have been performed with an anti-SARS-CoV-2 Spike antibody (figures E and F), a recombinant ACE2 protein (figure G) and an anti-ACE2 antibody (figure H).

For the three neutralizing assays, the HEK293 cells expressing ACE2, were transduced with a mix containing the Spike (SARS-CoV-2) pseudotyped lentivirus and different concentrations of the corresponding neutralizing compounds. The percentage of control transduction corresponds to the luciferase measured in the control wells with the ACE2 expressing cells and the virus, without any neutralizing compounds. These three independent experiments showed a lower transduction efficiency of the Spike (SARS-CoV-2) Pseudotyped Lentivirus (decrease in the luciferase expression), due to an inhibitory effect of the tested compounds.

In combination with ACE2 Lentivirus, this Spike (SARS-CoV-2) pseudotyped lentivirus can be used to study the mechanism of viral transduction, and easily screen for neutralizing compounds (antibodies – chemical compounds) of the SARS-CoV-2 Spike and ACE2 interaction, in a Biosafety Level 2 facility.

Bald Lentiviral Pseudovirion (Luciferase Reporter)

Bald LentivirusThe bald lentiviral pseudovirion was produced without envelope glycoproteins such as VSV-G or SARS-CoV-2 spike. It contains the firefly luciferase gene driven by a CMV promoter as the reporter. The bald lentiviral pseudovirion can serve as a negative control when studying virus entry initiated by specific interactions between virus particles and receptors.

 

 

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