WT‐RCMV‐E has previously been described (Ettinger et al, 2012 (link)). ΔE27‐RCMV‐E was generated by CRISPR/Cas9‐mediated genome editing. REF were transduced with a mixture of four CRISPR/Cas9 lentiviral vectors carrying sgRNAs that target viral genome up and downstream of the E27 gene (see Table EV3). Successfully transduced cells were selected with 2.5 μg/ml puromycin prior to infection with WT‐RCMV‐E. This resulted in an E27‐knockout virus (ΔE27) with a complete deletion of the 1970‐bp E27 gene. E27 deletion was verified by PCR and sequencing. Mutant virus was screened by limiting dilution and was double plaque‐purified. WT‐MCMV was reconstituted from the BAC described in (Jordan et al, 2011 (link)). ΔM27‐MCMV has been described previously (Le‐Trilling et al, 2018 (link)). The M27AxAxxA mutant was generated according to previously published procedures (Wagner et al, 2002 (link); Tischer et al, 2006 (link)) using the primers MCMV‐M27‐QC1‐3_1 and MCMV‐M27‐QC1‐3_2 (Table EV4). For the construction of the ΔM27‐E27HA virus, a DNA fragment harbouring one frt site, the ZeoR gene, the EF1 promoter and E27HA was introduced into the ΔM27‐MCMV‐BAC (harbouring an frt site instead of the M27) by FLP‐mediated homologous recombination in E. coli DH10B using the FLP‐expressing plasmid pCP20. Correct mutagenesis was confirmed by restriction digest analysis, Southern blot and PCR analysis. MCMV mutants were reconstituted by transfection of BAC DNA into permissive cells. Viral titres were determined by standard plaque titration on primary MEF, REF or MNC.