HeLa or D17 cells were seeded in 24-well plates (Corning) at densities of 2.5x104 cells/well and 2x104 cells/well, respectively. Cells were infected 24 h later, with WT and mutant VLPs normalised on their RT activity and incubated at 37°C for 72 h. Cells were then lysed in Tropix Lysis buffer (Thermo Fisher Scientific) and frozen at -20°C. To measure LacZ activity, cell lysate was mixed with Tropix galactostar reaction mixture (Thermo Fisher Scientific) and luminescence was measured for 1 h at 10 min intervals on a Tecan Safire plate reader.
Absolute infectious titres of VLPs for microscopy assays were determined by X-gal staining of infected HeLa cells. HeLa cells were seeded in 12-well plates (Corning) at a density of 5x104 cells/well and infected 24 h later with a 10-fold dilution series of VLPs by spinoculation (1600 g, 2 h, 16°C). Cells were then incubated at 37°C for 30 min prior to replacement of media with warm serum-supplemented DMEM. After 72 h, cells were washed in PBS and fixed for 10 min with 2% formaldehyde and 0.2% glutaraldehyde. Staining was performed overnight at 37°C in PBS with 0.4 mg/ml X-gal (5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (Sigma), 4 mM K3Fe(CN)6 (Sigma), 4mM K4Fe(CN)6ˑ3H2O (Sigma), 2mM MgCl2 (Thermo). The number of blue LacZ-expressing colonies were counted using a light microscope (Olympus) and the viral titre calculated.
Absolute infectious titres of VLPs for microscopy assays were determined by X-gal staining of infected HeLa cells. HeLa cells were seeded in 12-well plates (Corning) at a density of 5x104 cells/well and infected 24 h later with a 10-fold dilution series of VLPs by spinoculation (1600 g, 2 h, 16°C). Cells were then incubated at 37°C for 30 min prior to replacement of media with warm serum-supplemented DMEM. After 72 h, cells were washed in PBS and fixed for 10 min with 2% formaldehyde and 0.2% glutaraldehyde. Staining was performed overnight at 37°C in PBS with 0.4 mg/ml X-gal (5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (Sigma), 4 mM K3Fe(CN)6 (Sigma), 4mM K4Fe(CN)6ˑ3H2O (Sigma), 2mM MgCl2 (Thermo). The number of blue LacZ-expressing colonies were counted using a light microscope (Olympus) and the viral titre calculated.