A pseudovirus bearing CoV S protein or VSV-G protein and a defective HIV-1 genome that expresses luciferase as reporter was produced in 293 T cells, as previously described (27 (link)), and its titer was quantitated by using HIV-1 p24 ELISA. The pseudovirus was then used to infect target Huh-7 cells (or ACE2/293 T cells for pseudotyped SARS-CoV) (10 4 per well in 96-well plates) in the presence or absence of the test peptide at the indicated concentration. Twelve hours after infection, culture medium was refreshed and then incubated for an additional 48 hours, followed by washing cells with PBS, lysing cells with lysis reagent (Promega), and transferring the cell lysates to 96-well Costar flat-bottom luminometer plates (Corning Costar) for the detection of relative light units using the Firefly Luciferase Assay Kit (Promega) and an Ultra 384 luminometer (Tecan).
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Pseudovirus-based SARS-CoV-2 Infection Assay
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Corresponding Organization : Scripps Research Institute
Other organizations : The University of Texas Medical Branch at Galveston, Jazan University, National Institute for Viral Disease Control and Prevention
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Variable analysis
independent variables
- Test peptide at the indicated concentration
- Relative light units (luciferase activity)
- Huh-7 cells (or ACE2/293 T cells for pseudotyped SARS-CoV) (10^4 per well in 96-well plates)
- Culture medium refreshed 12 hours after infection and then incubated for an additional 48 hours
- Cells washed with PBS and lysed with lysis reagent (Promega)
- Cell lysates transferred to 96-well Costar flat-bottom luminometer plates (Corning Costar) for the detection of relative light units using the Firefly Luciferase Assay Kit (Promega) and an Ultra 384 luminometer (Tecan)
- Pseudovirus bearing CoV S protein or VSV-G protein and a defective HIV-1 genome that expresses luciferase as reporter, produced in 293 T cells and quantitated by using HIV-1 p24 ELISA
- Absence of the test peptide
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