Spike (B.1.621, Mu Variant) (SARS-CoV-2) Pseudotyped Lentivirus (Luc Reporter)

The pandemic coronavirus disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) . As the first step of the viral replication, the virus attaches to the host cell surface before entering the cell. The viral Spike protein recognizes and attaches to the Angiotensin-Converting Enzyme 2 (ACE2) receptor found on the surface of type I and II pneumocytes, endothelial cells, and ciliated bronchial epithelial cells. Drugs targeting the interaction between the Spike protein of SARS-CoV-2 and ACE2 may offer protection against the viral infection. A variant called B.1.621 (also known as the Mu Variant) was first identified in Columbia in early 2021. This variant has a number of mutations that may increase morbidity and mortality and allow the virus to spread more easily and quickly than other variants.The Spike (B.1.621, Mu Variant) (SARS-CoV-2) Pseudotyped Lentiviruses were produced with SARS-CoV-2 B.1.621 Variant Spike (Genbank Accession #QHD43416.1 with B.1.621 mutations; see below for details) as the envelope glycoproteins instead of the commonly used VSV-G.  These pseudovirions contain the firefly luciferase gene driven by a CMV promoter (Figure 1), therefore, the spike-mediated cell entry can be measured via luciferase activity. The Spike (B.1.621, Mu Variant) (SARS-CoV-2) pseudotyped lentivirus can be used to measure the activity of neutralizing antibody against the B.1.621 variant in a Biosafety Level 2 facility.Spike interacts with host cells by binding to membrane receptor ACE2 (angiotensin converting enzyme 2) . Based on experiments performed by scientists at BPS Bioscience, we know that the wild-type SARS-CoV-2 spike pseudotyped lentiviruses transduce the following cells with great efficiency:  ACE2-HEK293 cells (BPS Bioscience #79951), ACE2-CHO cells (BPS Bioscience #79959), ACE2-HeLa cells (BPS Bioscience #79958) . They also efficiently transduce TMPRSS2-Vero E6 cells (BPS Bioscience #78081), which express high endogenous levels of ACE2 and were stably transfected with human serine protease TMPRSS2  required for the priming of Spike and fusion of the virion with the plasma membrane. By contrast, it has been shown by others that SARS-CoV-2 spike pseudotyped lentiviruses do not transduce parental Calu3 and Vero E6 cells very well [Neerukondaet al. 2021, PlosOne PMID:33690649; Tandonet al. 2020, Scientific Reports PMID:33154514; Condor Capchaet al.2021, Front. Cardiovasc. Med. PMID:33521067; Pisilet al. 2021, Pathogens PMID:33540924].SARS-CoV-2 variant pseudoviruses have been validated using ACE2-HEK293 cells but have not been tested in other cells.As recommended in our protocol, 5 µl of virus/well in a 96-well plate provides a sufficient signal-to-noise ratio to perform inhibition studies. The amount of virus added to the cells can also be scaled down according to the user’s need.Spike Mutations in B.1.621 Variant:T95IY144SY145NR346KE484KN501YD614GP681HD950NFigure 1. Schematic of the Luciferase Reporter in SARS-CoV-2 Spike (B.1.621 Variant) Pseudovirion