Rapid, reliable, and reproducible cell fusion assay to quantify SARS-Cov-2 spike interaction with hACE2

Abstract

COVID-19 is a global crisis of unimagined dimensions. Currently, Remedesivir is only fully licensed FDA therapeutic. A major target of the vaccine effort is the SARS-CoV-2 spike-hACE2 interaction, and assessment of efficacy relies on time consuming neutralization assay. Here, we developed a cell fusion assay based upon spike-hACE2 interaction. The system was tested by transient co-transfection of 293T cells, which demonstrated good correlation with standard spike pseudotyping for inhibition by sera and biologics. Then established stable cell lines were very well behaved and gave even better correlation with pseudotyping results, after a short, overnight co-incubation. Results with the stable cell fusion assay also correlated well with those of a live virus assay. In summary we have established a rapid, reliable, and reproducible cell fusion assay that will serve to complement the other neutralization assays currently in use, is easy to implement in most laboratories, and may serve as the basis for high throughput screens to identify inhibitors of SARS-CoV-2 virus-cell binding and entry.

The FDA-Approved Drug Cobicistat Synergizes with Remdesivir To Inhibit SARS-CoV-2 Replication In Vitro and Decreases Viral Titers and Disease Progression in Syrian Hamsters

“…(E) Effect of cobicistat treatment on S-glycoprotein-mediated fusion. TZM-bl cells stably expressing the S-glycoprotein were incubated with different concentrations of cobicistat for 1 h and mixed with cells stably expressing human ACE2 ( 40 ). Cell fusion was assessed by measuring firefly luciferase activity after 24 h. RLU, relative light units.…”

“…Finally, to obtain a specific and quantitative estimate of the impact of cobicistat on the S-glycoprotein–ACE2 interaction, we tested the effect of this drug using a previously validated fusion assay ( 40 ) based on cell lines stably transfected with the S-glycoprotein and human ACE2. The results showed that cobicistat can inhibit S-glycoprotein fusion with an IC 50 of 3.8 μM ( Fig.…”

“…The effect of cobicistat on the interaction between the spike protein and human ACE2 was evaluated using a previously described fusion assay ( 40 ). Briefly, TZM-bl cells expressing the spike protein were incubated with 4-fold serial dilutions of cobicistat for 1 h. Target cells expressing the human ACE2 (h-ACE2) and the HIV-1 Tat protein were then mixed 1:1, and firefly luciferase activity was measured after 24 h.…”

Computational Design of Potent D-Peptide Inhibitors of SARS-CoV-2

“…A previous report showed that LCB1, a miniprotein SARS-CoV-2 inhibitor, can effectively inhibit cell fusion by co-incubating with SARS-CoV-2 at −1 and 0 h before adding the mix to Vero cells. 38 Here, we mixed different concentrations of the D-peptides with 100 TCID 50 of the SARS-CoV-2 isolates for 30 min at 37 °C before incubating the mix with the cells. Therefore, we did not expect to see a different mechanism of action for our designs if we preincubated these peptides with the virus during a similar time range.…”

Brilacidin, a COVID‐19 drug candidate, demonstrates broad‐spectrum antiviral activity against human coronaviruses OC43, 229E, and NL63 through targeting both the virus and the host cell

“…To delineate whether brilacidin blocks SARS‐CoV‐2 viral entry, we generated pseudotyped HIV‐1‐derived lentiviral particles with SARS‐CoV‐2 spike protein, 35 which is widely used to study spike‐mediated viral entry into host cells in biosafety Level 2 facilities. 40 , 41 Brilacidin was tested in SARS‐CoV‐2 pseudovirus entry assay in several ACE2‐expressing cell lines including Vero C1008, Calu‐3, Huh‐7, Caco‐2, and 293T‐ACE2. Vero C1008 and 293T‐ACE2 express minimal levels of transmembrane serine proteinase 2 (TMPRSS2), therefore the SARS‐CoV‐2 virus enters into these cell lines mainly through endocytosis and relies on endosomal cathepsin L for viral spike protein activation.…”