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. 2023 Oct 17;24(12):e56327. doi: 10.15252/embr.202256327

Figure 2. CNOT1 and CNOT3 promote viral gene expression late in HCMV infection.

Figure 2

  1. Immunoblot analysis of lysates from NHDF cells transfected with the indicated siRNAs and infected with HCMV AD169 at MOI = 3, collected at 72 HPI. Lysates were probed for representative viral proteins from each temporal expression class, select ISGs (MDA5, ISG15, PKR) and activated T446‐phosphorylated PKR (PKRph) and GAPDH.
  2. RT‐qPCR analysis for viral mRNAs from RNA isolated from cells treated and infected as in (A). Protein names, where different from gene names, indicated in brackets. Mean ± SEM (n = 6 biological replicates) is plotted with statistical significance established by ANOVA test with Dunnett multiple comparison correction compared with control siRNA; (**) P < 0.002, (***) P < 0.001.
  3. Immunoblot analysis of lysates from NHDF cells transfected with control or CNOT1 siRNAs and infected with HCMV AD169 at MOI = 3 collected at the indicated times post infection. Lysates were probed for representative viral proteins indicated, CNOT1 to confirm knockdown and a cellular loading control (GAPDH).
  4. NHDFs transfected with control or CNOT1‐specific siRNAs were infected with HCMV AD169 at MOI = 3, as in (A). At 72 hpi, total DNA was collected and HCMV DNA abundance was quantified by qPCR. Untransfected cells were treated in parallel with viral DNA polymerase inhibitor PAA after virus inoculation as a control. Mean ± SEM (n = 3 biological replicates) is plotted with statistical significance established by ANOVA test with Dunnett multiple comparison correction compared with control siRNA; (**) P < 0.002, (***) P < 0.001.

Source data are available online for this figure.