Herpesviruses persist indefinitely in their host through complex and poorly defined interactions that mediate latent, chronic or productive states of infection. Human cytomegalovirus (CMV or HCMV), a ubiquitous β-herpesvirus, coordinates the expression of two viral genes, UL135 and UL138, which have opposing roles in regulating viral replication. UL135 promotes reactivation from latency and virus replication, in part, by overcoming replication-suppressive effects of UL138. The mechanism by which UL135 and UL138 oppose one another is not known. We identified viral and host proteins interacting with UL138 protein (pUL138) to begin to define the mechanisms by which pUL135 and pUL138 function. We show that pUL135 and pUL138 regulate the viral cycle by targeting that same receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR). EGFR is a major homeostatic regulator involved in cellular proliferation, differentiation, and survival, making it an ideal target for viral manipulation during infection. pUL135 promotes internalization and turnover of EGFR from the cell surface, whereas pUL138 preserves surface expression and activation of EGFR. We show that activated EGFR is sequestered within the infection-induced, juxtanuclear viral assembly compartment and is unresponsive to stress. Intriguingly, these findings suggest that CMV insulates active EGFR in the cell and that pUL135 and pUL138 function to fine-tune EGFR levels at the cell surface to allow the infected cell to respond to extracellular cues. Consistent with the role of pUL135 in promoting replication, inhibition of EGFR or the downstream phosphoinositide 3-kinase (PI3K) favors reactivation from latency and replication. We propose a model whereby pUL135 and pUL138 together with EGFR comprise a molecular switch that regulates states of latency and replication in HCMV infection by regulating EGFR trafficking to fine tune EGFR signaling.

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.

Human cytomegalovirus, HCMV, is a betaherpesvirus that establishes a lifelong latent infection in its host that is marked by recurrent episodes of reactivation. The molecular mechanisms by which the virus and host regulate entry into and exit from latency remain poorly understood. We have previously reported that is critical for reactivation, functioning in part by overcoming suppressive effects of the latency determinant We have demonstrated a role for in diminishing cell surface levels and targeting epidermal growth factor receptor (EGFR) for turnover. The attenuation of EGFR signaling promotes HCMV reactivation in combination with cellular differentiation. In this study, we sought to define the mechanisms by which functions in regulating EGFR turnover and viral reactivation. Screens to identify proteins interacting with pUL135 identified two host adaptor proteins, CIN85 and Abi-1, with overlapping activities in regulating EGFR levels in the cell. We mapped the amino acids in pUL135 necessary for interaction with Abi-1 and CIN85 and generated recombinant viruses expressing variants of pUL135 that do not interact with CIN85 or Abi-1. These recombinant viruses replicate in fibroblasts but are defective for reactivation in an experimental model for latency using primary CD34 hematopoietic progenitor cells (HPCs). These variants have altered trafficking of EGFR and are defective in targeting EGFR for turnover. These studies demonstrate a requirement for pUL135 interactions with Abi-1 and CIN85 for regulation of EGFR and mechanistically link the regulation of EGFR to reactivation. Human cytomegalovirus (HCMV) establishes a lifelong latent infection in the human host. While the infection is typically asymptomatic in healthy individuals, HCMV infection poses life-threatening disease risk in immunocompromised individuals and is the leading cause of birth defects. Understanding how HCMV controls the lifelong latent infection and reactivation of replication from latency is critical to developing strategies to control HCMV disease. Here, we identify the host factors targeted by a viral protein that is required for reactivation. We define the importance of this virus-host interaction in reactivation from latency, providing new insights into the molecular underpinnings of HCMV latency and reactivation.

The maintenance of cell surface proteins is critical to the ability of a cell to sense and respond to information in its environment. As such, modulation of cell surface composition and receptor trafficking is a potentially important target of control in virus infection. Sorting endosomes (SEs) are control stations regulating the recycling or degradation of internalized plasma membrane proteins. Here we report that human cytomegalovirus (HCMV), a ubiquitous betaherpesvirus, alters the fate of internalized clathrin-independent endocytosis (CIE) cargo proteins, retaining them in virally reprogrammed SEs. We show that the small G protein ARF6 (ADP ribosylation factor 6), a regulator of CIE trafficking, is highly associated with SE membranes relative to uninfected cells. Combined with the observation of accumulated CIE cargo at the SE, these results suggest that infection diminishes the egress of ARF6 and its cargo from the SE. Expression of ubiquitin-specific protease 6 (USP6), also known as TRE17, was sufficient to restore ARF6 and some ARF6 cargo trafficking to the cell surface in infected cells. The USP activity of TRE17 was required to rescue both ARF6 and associated cargo from SE retention in infection. The finding that TRE17 expression does not rescue the trafficking of all CIE cargos retained at SEs in infection suggests that HCMV hijacks the normal sorting machinery and selectively sorts specific cargos into endocytic microdomains that are subject to alternative sorting fates.

Epithelial-mesenchymal transition (EMT) is an evolutionarily conserved process during which cells lose epithelial characteristics and gain a migratory phenotype. Although downregulation of epithelial cadherins by Snail and other transcriptional repressors is generally considered a prerequisite for EMT, recent studies have challenged this view. Here we investigate the relationship between E-cadherin and P-cadherin expression and localization, Snail function and EMT during gastrulation in chicken embryos. Expression analyses show that while E-cadherin transcripts are detected in the epiblast but not in the primitive streak or mesoderm, P-cadherin mRNA and protein are present in the epiblast, primitive and mesoderm. Antibodies that specifically recognize E-cadherin are not presently available. During EMT, P-cadherin relocalizes from the lateral surfaces of epithelial epiblast cells to a circumferential distribution in emerging mesodermal cells. Cells electroporated with an E-cadherin expression construct undergo EMT and migrate into the mesoderm. An examination of Snail function showed that reduction of Slug (SNAI2) protein levels using a morpholino fails to inhibit EMT, and expression of human or chicken Snail in epiblast cells fails to induce EMT. In contrast, cells expressing the Rho inhibitor peptide C3 rapidly exit the epiblast without activating Slug or the mesoderm marker N-cadherin. Together, these experiments show that epiblast cells undergo EMT while retaining P-cadherin, and raise questions about the mechanisms of EMT regulation during avian gastrulation.