Fig. 3: Effect of PAD2 and PAD4 gene knockout on HCMV replication.

a Knockout (KO) gene variants in HFFs for PAD2 (PAD2 KO) and PAD4 (PAD4 KO) were generated using CRISPR/Cas9 technology. The efficiency of PAD2 and PAD4 protein depletion at 48 hpi was assessed by immunoblotting using antibodies against PAD2, PAD4, or α-tubulin (α-TUB), for equal loading. An anti-UL44 antibody was used to verify HCMV infection. The western blot and relative densitometric analysis are representative of three independent experiments. Values are expressed as fold change in PAD2 and 4 expression normalized to α-tubulin (α-TUB). b HFFs KO cells were infected with HCMV at an MOI of 0.1 PFU/cell. Viral supernatants were collected at the indicated time points and analyzed by standard plaque assay. Values are expressed as means ± SEM of three independent experiments (WT vs. PAD2 KO P < 0.001, WT vs. PAD4 KO P < 0.001, one-way ANOVA followed by Bonferroni’s post test). c To determine the number of viral DNA genomes in HCMV-infected HFFs KO cells (MOI 0.1), viral DNA was isolated at 144 hpi and analyzed by qPCR with primers amplifying a segment of the IE1 gene. GAPDH was used to normalize HCMV genome counts. Values are expressed as mean ± SEM of three independent experiments (WT vs. PAD2 KO P < 0.001, WT vs. PAD4 KO P < 0.001, one-way ANOVA followed by Bonferroni’s post test). Data are shown as the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.