EGR1 transcriptional control of human cytomegalovirus latency

ABSTRACT Sustained phosphotinositide3-kinase (PI3K) signaling is critical to the maintenance of herpesvirus latency. We have previously shown that the beta-herpesvirus, human cytomegalovirus (CMV), regulates epidermal growth factor receptor (EGFR), upstream of PI3K, to control states of latency and reactivation. Inhibition of EGFR signaling enhances CMV reactivation from latency and increases viral replication, but the mechanisms by which EGFR impacts replication and latency is not known. We demonstrate that HCMV downregulates MEK/ERK and AKT phosphorylation, but not STAT3 or PLCγ for productive replication. Similarly, inhibition of either MEK/ERK or PI3K/AKT, but not STAT or PLCγ, pathways increases viral reactivation from latently infected CD34 + hematopoietic progenitor cells (HPCs), defining a role for these pathways in latency. We hypothesized that CMV modulation of EGFR signaling might impact viral transcription. Indeed, EGF-stimulation increased expression of the UL138 latency gene, but not immediate early or early viral genes, suggesting that EGFR signaling promotes latent gene expression. The early growth response-1 (EGR1) transcription factor is induced downstream of EGFR signaling through both PI3K/AKT and MEK/ERK pathways. EGR1 expression is important for the maintenance of HPC stemness and its downregulation drives HPC differentiation and mobilization. We demonstrate that EGR1 binds upstream of UL138 and is sufficient to promote UL138 expression. Further, disruption of EGR1 binding upstream of UL138 prevented CMV from establishing a latent infection in CD34 + HPCs. Our results indicate a model whereby UL138 modulation of EGFR signaling feeds back to promote UL138 expression and suppression of replication to establish or maintain viral quiescence. AUTHOR SUMMARY CMV regulates EGFR signaling to balance states of viral latency and replication. CMV blocks downstream PI3K/AKT and MEK/ERK signaling pathways through down-regulation of EGFR at the plasma membrane. PI3K/AKT and MEK/ERK signaling increases expression of the EGR1 transcription factor that is necessary for the maintenance of stem cell stemness. A decrease in EGR1 expression promotes HPC mobilization to the periphery and differentiation, a known stimulus for CMV reactivation. We identified functional EGR1 binding sites upstream of the UL138 gene and EGR-1 binding stimulates UL138 expression. Additionally, down-regulation of EGR1 by CMV miR-US22 decreases UL138 expression emphasizing the importance of this transcription factor in expression of this latency gene. Lastly, we demonstrate that a CMV mutant virus lacking an upstream EGR1 binding site is unable to establish latency in CD34 + HPCs. This study defines one mechanism by which EGFR signaling impacts viral gene expression to promote CMV latency.