Lipofectamine 3000

For pseudoviruses construction, spike genes from strain Wuhan-Hu-1 (GenBank: MN908947) were codon-optimized for human cells and cloned into eukaryotic expression plasmid pcDNA3.1 to generate the envelope recombinant plasmids pcDNA3.1.S2.
The pseudoviruses were produced and titrated using methods similar to Rift valley fever pseudovirus, as described previously [19 (link),20 (link)]. For this VSV pseudovirus system, the backbone was provided by VSV G pseudotyped virus (G*ΔG-VSV) that packages expression cassettes for firefly luciferase instead of VSV-G in the VSV genome. Briefly, 293T cells were transfected with pcDNA3.1.S2 (30 μg for a T75 flask) using Lipofectamine 3000 (Invitrogen, L3000015) following the manufacturer’s instruction. Twenty-four hours later, the transfected cells were infected with G*ΔG-VSV with a multiplicity of four. Two hours after infection, cells were washed with PBS three times, and then new complete culture medium was added. Twenty-four hours post infection, SARS-CoV-2 pseudoviruses containing culture supernatants were harvested, filtered (0.45-μm pore size, Millipore, SLHP033RB) and stored at −70°C in 2-ml aliquots until use. The 50% tissue culture infectious dose (TCID₅₀) of SARS-CoV-2 pseudovirus was determined using a single-use aliquot from the pseudovirus bank; all stocks were used only once to avoid inconsistencies that could have resulted from repeated freezing-thawing cycles. For titration of the SARS-CoV-2 pseudovirus, a 2-fold initial dilution was made in hexaplicate wells of 96-well culture plates followed by serial 3-fold dilutions (nine dilutions in total). The last column served as the cell control without the addition of pseudovirus. Then, the 96-well plates were seeded with trypsin-treated mammalian cells adjusted to a pre-defined concentration. After 24 h incubation in a 5% CO₂ environment at 37°C, the culture supernatant was aspirated gently to leave 100 μl in each well; then, 100 μl of luciferase substrate (Perkinelmer, 6066769) was added to each well. Two min after incubation at room temperature, 150 μl of lysate was transferred to white solid 96-well plates for the detection of luminescence using a microplate luminometer (PerkinElmer, Ensight). The positive well was determined as ten-fold relative luminescence unit (RLU) values higher than the cell background. The 50% tissue culture infectious dose (TCID₅₀) was calculated using the Reed–Muench method, as described previously [13 (link),18 (link),19 (link)].

HeLa cells transiently expressing ACE2 were prepared using Lipofectamine 3000 (Thermo Fisher Scientific) in a 96-well plate; mock-transfected cells were used as controls. 2019-nCoV grown in Vero E6 cells was used for infection at a MOI of 0.5. APN and DPP4 were analysed in the same way. The inoculum was removed after absorption for 1 h and washed twice with PBS and supplemented with medium. At 24 h after infection, cells were washed with PBS and fixed with 4% formaldehyde in PBS (pH 7.4) for 20 min at room temperature. ACE2 expression was detected using a mouse anti-S tag monoclonal antibody and a FITC-labelled goat anti-mouse IgG H&L (Abcam, ab96879). Viral replication was detected using a rabbit antibody against the Rp3 N protein (generated in-house, 1:1,000) and a Cy3-conjugated goat anti-rabbit IgG (1:200, Abcam, ab6939). Nuclei were stained with DAPI (Beyotime). Staining patterns were examined using confocal microscopy on a FV1200 microscope (Olympus).