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.