Fig 4. US28 attenuates Akt signaling to establish quiescence.

(A to C) Monocytes were infected with WT or US28Δ HCMV (MOI = 1). After 30 min to allow uninterrupted viral entry, monocytes were treated with the EGFR inhibitor AG1478 (5 μM), the pan-PI3K inhibitor LY294002 (25 μM) or the Akt inhibitor MK2206 (10 μM). After 24 h, IE1 protein and UL123 transcript levels were measured by Western blot (A, B) and RT-qPCR analysis (C), respectively. RT-qPCR data are means ± SEM from at least 3 biological replicates per group. (D to F) Phosphorylation of Akt at Ser⁴⁷³ and Thr³⁰⁸ and total Akt were detected by Western blot in monocytes after 30 min of infection (D). β-actin was used as a loading control. Levels of phosphorylated or total Akt were normalized to actin (E, F). The phosphorylation ratios of Ser⁴⁷³ or Thr³⁰⁸ to total Akt were determined with WT infection set to 1 (E). Total Akt abundance is shown relative to WT infection, which was set to 1 (F). Western blots and densitometry are representative of at least 3 biological replicates per group. *P<0.05, **P < 0.005, ***P < 0.0005 by one-way ANOVA with Tukey’s HSD post hoc test.