0.45 μm pore size filter

The CEM cells with the different LPAP mutants were generated by lentiviral transduction of CEM98 cells. For initiation of HIV-1 infection, HEK293T cells were cotransfected with 0.6 μg of HIV-1 packaging plasmid pCMVΔ8.2R (Addgene), 0.9 μg of pUCHR LPAP IRES GFP transfer vector, and 0.15 μg of pCMV-VSVG plasmid (Addgene) expressing Env G from vesicular stomatitis virus, using Lipofectamine 2000 (Invitrogene). The next day the culture medium was replaced, and cells were grown for another 24 h. Supernatant from the 6-cm dish with transfected HEK293T cells was harvested and clarified through a 0.45 μm pore size filter (Corning). Virus-like particles (VLPs) in the supernatant were concentrated by centrifugation at 100,000 g for 2.5 h and resuspended in 0.5 mL of fresh RPMI culture medium. Freshly prepared VLPs in a volume of 1 mL were added to 1 × 10⁵ CEM cells. Cells were grown for 4 days and sorted by GFP expression.

Pseudotyped ΔG-luciferase rVSV with SARS-CoV-2 glycoprotein or SARS-CoV-1 glycoprotein was performed as similarly described in recent publication (2 (link)). In brief, 80% confluent BHK21 were transfected with SARS-CoV-2 or SARS-CoV-1 spike glycoprotein expression plasmid. Transfected cells were culture at 37°C with 7% CO₂ in reduced serum DMEM for 30 h. Next day, the transfection medium was removed, and cells were infected with pseudotyped ΔG-luciferase rVSV in serum and antibiotic free DMEM at an MOI of 3. After 4 h of infection, the cells were washed with 1× DPBS, reduced serum DMEM was added, and cells were further cultured for another 24 h. On the following day, pseudotyped viruses in the culture medium were collected and filtered using 0.45-μm-pore-size filter (Corning); the filtrate was ultracentrifuged with 20% sucrose cushion at 100,000g for 35 min at 4°C and reconstituted in PBS for experimental use. Cultured hACE2-A549 cells at an appropriate density were added in the medium with a dose of pseudotyped SARS-CoV-2 or pseudotyped SARS-CoV-1 and about 1 h later were added with a dose of Ex/Mv or vehicle in the medium. Cells were maintained under these conditions for 3 d before fixed or lysed for subsequent assays.

Murine leukemia virus pseudoparticles were generated using the method described previously [16 (link)]. 293T cells (American Type Culture Collection) were seeded in 10-cm dishes at 2 x 10⁶ cells/dish and were cultured in 8 mL DMEM with 10% FBS at 5% CO₂. On the following day, each dish of these cultures was transfected, using FuGENE6 transfection reagent (Roche Diagnostics), with a mixture consisting of plasmid DNAs corresponding to the gag-pol packaging vector (3.1 μg), the transfer vector (3.1 μg), and the HCV glycoprotein-encoding vector (1 μg). The HCV glycoprotein-encoding vector harbored DNA sequences encoding a segment of the HCV glycoprotein corresponding to amino acid (aa) 132–747 from the TH strain (genotype 1b), or aa 132–750 from the J6CF strain (genotype 2a). After 48 hours, pseudoparticle-containing supernatants were collected and passed through a 0.45-μm-pore-size filter (Corning, NY, USA). The collected pseudoparticles were stored at -80°C until use.