MLV-based pseudovirus assay

Murine Leukemia Virus (MLV) particles (plasmids of the MLV components kindly provided by Dr. Gary Whittaker at Cornell University and Drs. Catherine Chen and Wei Zheng at National Center for Advancing Translational Sciences, National Institutes of Health), pseudotyped with various SARS-CoV-2 S protein constructs, were generated in HEK 293T cells, following a protocol described previously for SARS-CoV (Chen et al., 2020; Millet and Whittaker, 2016). To enhance incorporation of S protein into the particles, the C-terminal 19 residues in the cytoplasmic tail of each S protein were deleted. To increase the cleavage between S1 and S2, 1.5 μg of the furin expression construct was added into the DNA mixture (20 μg) for MLV particle production. To prepare for infection, 7.5 × 10³ of HEK 293 cells, stably transfected with a full-length human ACE2 expression construct, in 15 μL culture medium were plated into a 384-well white-clear plate coated with poly-D-Lysine to enhance the cell attachment. On day 2, 15 μL of MLV pseudoviruses for each variant were added into each well pre-seeded with HEK293-ACE2 cells. The plate was centrifuged at 114 xg for 5 min at 12°C. After incubation of the pseudoviruses with the cells for a time period (10 min-8 hr), as indicated in the figures, the medium was removed and the cells were washed once with 1xDPBS. 30 μL of fresh medium was added back into each well. The cells were then incubated at 37°C for additional 40 hr. Luciferase activities were measured with Firefly Luciferase Assay Kit (CB-80552-010, Codex BioSolutions Inc).