Virus and virus titration
The recombinant HCMV (RCMV1111) used was derived by transfection of a BAC clone of HCMV strain Merlin, the genome of which is designated the reference HCMV sequence by the National Center for Biotechnology Information and was sequenced after 3 passages in vitro (Dolan et al., 2004;Stanton et al., 2010). Virus stocks were prepared from HFFF-TERTs as described previously (Nobre et al., 2019). Tissue culture supernatants were kept when a 100% cytopathic effect was observed, and were centrifuged to remove cell debris. Cell-free virus was pelleted from supernatant by centrifugation at 15,000 × g for 2 h and then resuspended in fresh DMEM. Residual debris was removed from the resulting virus stocks by centrifugation at 10,000 x g for 1 min. Virus titration was achieved by intracellularly staining HCMV IE1/2 in HFFF-TERTs that had been infected with serially diluted HCMV. Cells were harvested 24 h post-infection, fixed in 4% paraformaldehyde, permeabilised with ice-cold methanol, blocked with human TruStain FcX Fc receptor blocking solution (Biolegend) and then subjected to primary (anti-HCMV IE1/2, mouse monoclonal 6F8.2, Millipore) and secondary (anti-mouse IgG conjugated with Alexa Fluor 488, Thermo) antibody incubation. Data was acquired by FACSCalibur (BD biosciences) and analysed with FlowJo software (BD biosciences). The percentage of infected cells was determined by the percentage of IE1/2 positive cells, which was used to calculate the titre of virus stock.