PMC:7443692 / 68312-70536 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/7443692","sourcedb":"PMC","sourceid":"7443692","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7443692","text":"293T cells were transfected with an expression plasmid encoding SARS-CoV-2 Spike (pcDNAintron-SARS-2-SΔ19). To increase cell surface expression, the last 19 amino acids containing the Golgi retention signal were removed. Two SΔ19 constructs were compared, one started with Met1 and the other with Met2. Twenty-four h following transfection, cells were transduced with ppVSVΔG-VSV-G (particles that were pseudotyped with VSV-G in trans). One h following transduction cells were extensively washed and media was replaced. Supernatant containing particles were collected 12-24 h following transduction and cleared through centrifugation. Cleared supernatant was frozen at −80°C for future use. Target cells Vero E6 were seeded in 24-well plates (5x105 cells/mL) at a density of 80% coverage. The following day, ppVSV-SARS-2-S/GFP particles were transduced into target cells for 60 min, particles pseudotyped with VSV-G, Lassa virus GP, or no glycoprotein were included as controls. 24 h following transduction, transduced cells were released from the plate with trypsin, fixed with 4% formaldehyde, and GFP-positive virus-transduced cells were quantified using flow cytometry (Bectin Dickson BD-LSRII). To quantify the ability of various SARS-CoV-2 S mutants to mediate fusion, effector cells (HEK293T) were transiently transfected with the indicated pcDNAintron-SARS-2-S expression vector or measles virus H and F (Brindley et al., 2014). Effector cells were infected with MVA-T7 four h following transduction to produce the T7 polymerase (Paal et al., 2009). Target cells naturally expressing the receptor ACE2 (Vero) or ACE2 negative cells (HEK293T) were transfected with pTM1-luciferase, which encodes for firefly luciferase under the control of a T7 promoter (Brindley and Plemper, 2010). 24 h following transfection, the target cells were lifted and added to the effector cells at a 1:1 ratio. 4 h following co-cultivation, cells were washed, lysed and luciferase levels were quantified using Promega’s Steady-Glo substrate. To visualize cell-to-cell fusion, Vero cells were co-transfected with pGFP and the pcDNAintron-SARS-2-S constructs. 24 h following transfection, syncytia was visualized by fluorescence 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