Fig 10. Inhibition of EGR1 binding to site 1 disrupts CMV latency.

(A) CD34⁺ HPCs were infected with either WT TB40/E_GFP or TB40/E-ΔEGR1_{Site 1} mutant virus (MOI = 2). At 24 hpi, CD34⁺/GFP⁺ cells were sorted and seeded into long-term culture. After 10 days in culture, parallel populations of either mechanically lysed cells or whole cells were plated onto fibroblast monolayers in cytokine-rich media. 14 days later, GFP+ wells were scored and frequency of infectious centers was determined by extreme limited dilution analysis (reactivation). The mechanically lysed population defines the quantity of virus present prior to reactivation (pre-reactivation). The frequency was normalized to WT pre-reactivation and the average of three independent experiments is shown. Statistical significance was calculated by Two-Way ANOVA with Tukey’s correction and represented by asterisks (* p-value < 0.05). (B) At 5 dpi, RNA was isolated from WT- and ΔEGR1_Site1-infected CD34⁺ HPCS and transcripts encoding UL135 and UL138 was quantified by RT-qPCR. A ratio of UL138 to UL135 contain transcripts was calculated with Pfaffl.