Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency

Abstract

Sustained phosphotinositide3-kinase (PI3K) signaling is critical to the maintenance of alpha and beta 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. How signaling downstream of EGFR is regulated and how this impacts CMV infection and latency is not fully understood. We demonstrate that CMV downregulates EGFR early in the productive infection, which blunts the activation of EGFR and its downstream pathways in response to stimuli. However, CMV infection sustains basal levels of EGFR and downstream pathway activity in the context of latency in CD34+ hematopoietic progenitor cells (HPCs). Inhibition of MEK/ERK, STAT or PI3K/AKT pathways downstream of EGFR increases viral reactivation from latently infected CD34⁺ HPCs, defining a role for these pathways in latency. We hypothesized that CMV modulation of EGFR signaling might impact viral transcription important to latency. 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 the MEK/ERK pathway and is important for the maintenance of hematopoietic stemness. We demonstrate that EGR1 binds the viral genome upstream of UL138 and is sufficient to promote UL138 expression. Further, disruption of EGR1 binding upstream of UL138 prevents the establishment of latency in CD34⁺ HPCs. Our results indicate a model whereby UL138 modulation of EGFR signaling feeds back to promote UL138 gene expression and suppression of replication for latency. By this mechanism, the virus has hardwired itself into host cell biology to sense and respond to changes in homeostatic host cell signaling. Host signaling is important for regulating states of cytomegalovirus (CMV) replication and latency. We have shown that human cytomegalovirus regulates EGFR levels and trafficking and that sustained EGFR or downstream PI3K signaling is a requirement for viral latency. Changes in host signaling have the ability to alter viral and host gene expression to impact the outcome of infection. Here we show that EGFR signaling through MEK/ERK pathway induces the host EGR1 transcription factor that is highly expressed in hematopoietic stem cells and necessary for the maintenance of hematopoietic stemness. Downregulation of EGR1 promotes stem cell mobilization and differentiation, known stimuli for CMV reactivation. We identified functional EGR1 binding sites upstream of the UL138 CMV latency gene and EGR1 stimulated UL138 expression to reinforce the latent infection. Mutant viruses where the regulation of UL138 by EGR1 is disrupted are unable to establish latency in CD34⁺ HPCs. This study advances our understanding of how host signaling impacts decisions to enter into or exit from latency. The regulation of viral gene expression by host signaling allows the virus to sense and respond to changes in host stress or differentiation.