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.

Levels of K in nectar of onion (Allium cepa L.) varied from 3600-13,000 ppm, about 10 times higher than that of nectar from competing flora tested. An open-pollinated amphidiploid derived from the interspecific cross A. cepa X A. fistulosum L. was the only onion with nectar K level as low as that of competing flora and it was more attractive to honey bees than was the common onion, A. cepa. Addition of K salts to sugar solutions at the rate of 1500, 3000, 4500, 6000, or 7500 ppm K clearly reduced uptake of these solutions by honey bees foraging at artificial-flower feeders. Thus, it appears that the levels of K in onion nectar are high enough to reduce the attractiveness of onion flowers to honey bees. It is suggested that pollination of onion flowers might be improved if nectar K levels can be reduced, either through a program of selective breeding or by altering cultural practices. An alternative solution might be to provide pollinators that readily accept the high K level present in onion nectar. This might be accomplished by preconditioning honey bees to accept high K levels in sugar solutions before they are moved to the onion seed fields, by developing a strain of honey bees with a preference for onion (high K) nectar, or by utilizing other species of pollinators.

34Sustained phosphotinositide3-kinase (PI3K) signaling is critical to the maintenance of 35 herpesvirus latency. We have previously shown that the beta-herpesvirus, human 36 cytomegalovirus (CMV), regulates epidermal growth factor receptor (EGFR), upstream of PI3K, 37 to control states of latency and reactivation. Inhibition of EGFR signaling enhances CMV 38 reactivation from latency and increases viral replication, but the mechanisms by which EGFR 39 impacts replication and latency is not known. We demonstrate that HCMV downregulates 40 MEK/ERK and AKT phosphorylation, but not STAT3 or PLCγ for productive replication. 41Similarly, inhibition of either MEK/ERK or PI3K/AKT, but not STAT or PLCγ, pathways increases 42 viral reactivation from latently infected CD34 + hematopoietic progenitor cells (HPCs), defining a 43 role for these pathways in latency. We hypothesized that CMV modulation of EGFR signaling 44 might impact viral transcription. Indeed, EGF-stimulation increased expression of the UL138 45 latency gene, but not immediate early or early viral genes, suggesting that EGFR signaling 46 promotes latent gene expression. The early growth response-1 (EGR1) transcription factor is 47 induced downstream of EGFR signaling through both PI3K/AKT and MEK/ERK pathways. 48 EGR1 expression is important for the maintenance of HPC stemness and its downregulation 49 drives HPC differentiation and mobilization. We demonstrate that EGR1 binds upstream of 50 UL138 and is sufficient to promote UL138 expression. Further, disruption of EGR1 binding 51 upstream of UL138 prevented CMV from establishing a latent infection in CD34 + HPCs. Our 52 results indicate a model whereby UL138 modulation of EGFR signaling feeds back to promote 53 UL138 expression and suppression of replication to establish or maintain viral quiescence. 54 55 AUTHOR SUMMARY 56 Buehler et al. 3CMV regulates EGFR signaling to balance states of viral latency and replication. CMV blocks 57 downstream PI3K/AKT and MEK/ERK signaling pathways through down-regulation of EGFR at 58 the plasma membrane. PI3K/AKT and MEK/ERK signaling increases expression of the EGR1 59 transcription factor that is necessary for the maintenance of stem cell stemness. A decrease in 60 EGR1 expression promotes HPC mobilization to the periphery and differentiation, a known 61 stimulus for CMV reactivation. We identified functional EGR1 binding sites upstream of the 62 UL138 gene and EGR-1 binding stimulates UL138 expression. Additionally, down-regulation of 63 EGR1 by CMV miR-US22 decreases UL138 expression emphasizing the importance of this 64 transcription factor in expression of this latency gene. Lastly, we demonstrate that a CMV 65 mutant virus lacking an upstream EGR1 binding site is unable to establish latency in CD34 + 66HPCs. This study defines one mechanism by which EGFR signaling impacts viral gene 67 expression to promote CMV latency. 68 69 RESULTS 129CMV downregulates total and cell surface levels of EGFR. We previously demonstrated that 130 CMV modulates EGFR total and cell s...