Paul.xu: Created page with "Briefly, 293T cells (ATCC, Manassas, VA) were co-transfected with 20 μg of plasmid encoding Env-defective, luciferase-expressing HIV-1 (pNL4-3.luc.RE) and 20 μg of rMERS-CoV..."

2020-04-21T15:25:23Z

Created page with "Briefly, 293T cells (ATCC, Manassas, VA) were co-transfected with 20 μg of plasmid encoding Env-defective, luciferase-expressing HIV-1 (pNL4-3.luc.RE) and 20 μg of rMERS-CoV..."

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Briefly, 293T cells (ATCC, Manassas, VA) were co-transfected with 20 μg of plasmid encoding Env-defective, luciferase-expressing HIV-1 (pNL4-3.luc.RE) and 20 μg of rMERS-CoV-S plasmid, respectively, into a-T175 tissue culture flask using the calcium phosphate method. Cells were changed into fresh DMEM 8 h later. Supernatants were harvested 72 h post-transfection and used for single-cycle infection. The plasmids encoding vesicular stomatitis virus G protein (VSV-G-pcDNA3.1) and pcDNA3.1 vector were co-transfected with pNL4-3.luc.RE plasmid to generate VSV-G pseudovirus and pseudovirus without Env (Env-) as controls

HIV-1 p24 The p24 content in the produced MERS-CoV pseudovirus was quantified by ELISA, as previously described, with some modifications [10]. Briefly, ELISA plates were precoated with HIVIG (5 μg/ml) overnight at 4°C and blocked at 37°C for 2 h. Lysed pseudovirus was added to the plates and incubated at 37°C for 1 h. After washes, the plates were respectively incubated with anti-p24 mAb (183-H12-5C, 1:20) and then with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG at 37°C for 1 h. The substrate 3,30,5,50-tetramethylbenzidine (TMB) (Zymed, Carlsbad, CA) was added, and the reaction was stopped by 1 N H2SO4. The absorbance at 450 nm (A450) was measured by ELISA Plate Reader (Tecan, San Jose, CA).

Detection of MERS-CoV pseudovirus infectivity To detect infectivity, different target cells (104/well in 96-well plates) from various sources (Table 1) were respectively infected with MERS-CoV pseudovirus. Fresh DMEM was added 12 h post-infection, and RLU was measured 72 h later by Ultra 384 luminometer (Tecan).

Western blot Western blot was performed to detect HIV-1 p24 and MERS-CoV S protein in the generated pseudovirus, and to identify expression of DPP4 in cells susceptible to MERS-CoV pseudovirus as previously described with some modifications [10]. Briefly, lysed pseudovirus (200 ng/ml p24) or cell lysates (30 μg total proteins) were respectively separated by 10% Tris-glycine gels, which were then transferred to nitrocellulose membranes. After blocking with 5% non-fat milk in PBST overnight at 4°C, the blots were respectively incubated with anti-p24 (183-H12-5C, 1:50), anti-S of MERS-CoV (1:200) (prepared in our laboratory), and goat anti-DPP4 (1 μg/ml, R&D Systems, Minneapolis, MN), for 1 h at room temperature. After three washes, the blots were then incubated with HRP-conjugated anti-mouse IgG (for HIV-1 p24 and MERS-CoV S, 1:3,000, Invitrogen) or anti-goat IgG (for DPP4, 1 1:3,000, R&D Systems) for 1 h at room temperature. Signals were visualized with ECL Western blot substrate reagents and Amersham Hyperfilm (GE Healthcare).
[[Category:Virus]]

VSVG-Pseudovirus - Revision history

Paul.xu: Created page with "Briefly, 293T cells (ATCC, Manassas, VA) were co-transfected with 20 μg of plasmid encoding Env-defective, luciferase-expressing HIV-1 (pNL4-3.luc.RE) and 20 μg of rMERS-CoV..."