Opposing Regulation of the EGF Receptor: A Molecular Switch Controlling Cytomegalovirus Latency and Replication

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

HCMV Induced Alterations to Endocytic Sorting

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 beta herpesvirus, 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, a regulator of CIE trafficking, is highly associated with SE membranes, relative to uninfected cells. This finding suggests that ARF6 and CIE cargo egress from the SE is diminished by infection. Over expression of the ubiquitin specific protease (USP) 6, 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 is required to rescue both ARF6 and associated cargo from SE retention in infection. Intriguingly, TRE17 expression does not affect all CIE cargos retained at SEs in infection. Although TRE17 mediates the trafficking of internalized major histocompatibility complex type I (MHCI) to the cell surface in uninfected cells, MHCI is insensitive to TRE17-mediated trafficking in the context of HCMV infection. These findings demonstrate a reprogramming of endocytic trafficking by HCMV infection and suggests that HCMV hijacks the normal sorting machinery and selectively sorts specific cargos into endocytic micro-domains that are subject to alternate sorting fates

Hcmv Induced Alterations to Endocytic Sorting

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 beta herpesvirus, 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, a regulator of CIE trafficking, is highly associated with SE membranes, relative to uninfected cells. This finding suggests that ARF6 and CIE cargo egress from the SE is diminished by infection. Over expression of the ubiquitin specific protease (USP) 6, 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 is required to rescue both ARF6 and associated cargo from SE retention in infection. Intriguingly, TRE17 expression does not affect all CIE cargos retained at SEs in infection. Although TRE17 mediates the trafficking of internalized major histocompatibility complex type I (MHCI) to the cell surface in uninfected cells, MHCI is insensitive to TRE17-mediated trafficking in the context of HCMV infection. These findings demonstrate a reprogramming of endocytic trafficking by HCMV infection and suggests that HCMV hijacks the normal sorting machinery and selectively sorts specific cargos into endocytic micro-domains that are subject to alternate sorting fates

Virus Control of Trafficking from Sorting Endosomes

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. IMPORTANCE Cells maintain their surface composition, take up nutrients, and respond to their environment through the internalization and recycling of cargo at the cell surface through endocytic trafficking pathways. During infection with human cytomegalovirus (HCMV), host endocytic membranes are reorganized into a juxtanuclear structure associated with viral assembly and egress. Less appreciated is the effect of this reorganization on the trafficking of host proteins through the endocytic pathway. We show that HCMV retains internalized cargo and the effector of clathrin-independent endocytosis at sorting endosomes. The retention of some cargo, but not all, was reversed by overexpression of a ubiquitin-specific protease, TRE17. Our results demonstrate that HCMV induces profound reprogramming of endocytic trafficking and influences cargo sorting decisions. Further, our work suggests the presence of a novel ubiquitin-regulated checkpoint for the recycling of cargo from sorting endosome. These findings have important implications for host signaling and immune pathways in the context of HCMV infection.Public Health Service from the National Institute of Allergy and Infectious Disease [AI079059, AI131598]; National Institute of Diabetes and Digestion and Kidney Diseases [DK109701A1]; National Heart Lung and Blood Institute at NIH [HL006060-08]; Cytometry Shared Resource, University of Arizona Cancer Center [P30CA023074]Open access journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

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

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.National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R01 AI21640, P01 AI127335, R01 AI079059]; National Cancer Institute institutional T32 training grant [T32CA009213-36]; American Cancer Society Post-Doctoral Research Fellowship [129842-PF-16-212-01-TBE]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Gastrulation EMT Is Independent of P-Cadherin Downregulation - Fig 2

<p>Co localization of E-cad and P-cad in using a P-cad-specific antibody prepared against a peptide from the extracellular domain of P-cad that shares only three amino acids with the corresponding sequence in E-cad (A), and a commercially obtained antibody raised against the E-cad intracellular domain that recognizes both P-cad and E-cad. (B-D) Transverse section through the mid streak region of a HH stage 4 embryos, showing immunolocalization E-cad and/or P-cad (B), or P-cad (C), or both (D).</p

Location of SNAI2 morpholino and control morpholino containing cells in transverse sections of embryos.

<p>A fluorescein-labeled morpholino targeting the initiation of translation of the <i>SNAI2</i> mRNA, or a five base pair mismatch control morpholino, were electroporated into the epiblast of HH stage 3–4 embryos. (A-B) The same microscopic field visualizing cells containing the control morpholino (green) and Slug (red) in (A), or Slug (B). Almost all cells containing the control morpholino also express Slug protein (white arrows). (C-D) The same microscopic field visualizing cells containing the SNAI2 morpholino and Slug (C), or Slug (D). Most cells in the primitive streak containing SNAI2 morpholinos were Slug negative (white arrows). (E,F) Higher magnification views showing cells containing the SNAI2 morpholino, and lacking detectable Slug protein, undergoing EMT (arrows).</p

C3 induces EMT without activating Slug or upregulating N-cad.

<p>(A,B) Transverse sections showing the same microscopic field four hours following electroporation with the C3 expression vector. While cells expressing C3 (green) in the primitive streak region within the domain of normal Slug expression were also Slug-positive (arrowheads in A, B), C3 expressing cells in more lateral regions that were exiting the epiblast or that had migrated into the mesoderm failed to express detectable levels of Slug (arrows in A, B). (C, D) C3 expressing cells that had undergone EMT and migrated into the mesoderm failed to express N-cad.</p

Localization of E-cad and/or P-cad and N-cad in transverse sections of a HH stage 3–4 chicken embryos.

<p>(A) Brightfield image of a HH stage 3 embryo following processing for immunofluorescence localization of E-cad and/or P-cad and N-cad, showing the location of images in (B-D’). Expression of E-cad and/or P-cad (B,C,D), or E-cad and/or P-cad plus N-cad (B’, C’, D’) in transverse sections. (E, F) Higher magnification images of boxed area in (D’), showing the localization of E-cad and/or P-cad (E) and N-cad (F) as cells transition from epiblast to mesoderm. E-cad and/or P-cad are retained on the surface of cells during EMT and after cells emerge into the mesoderm. Arrows point to rounded cells in the ventral streak showing robust detection of E-cad and/or P-cad at the cell periphery. (G, H) Co localization of E-cad and/or P-cad and N-cad in transverse sections of HH stage 4 embryos, showing their heterogeneous expression between mesoderm cells.</p

Protein localization in transverse sections of gastrulating chicken and mouse embryos.

<p>(A-C) Localization of E-cad and/or P-cad, p120-catenin, GM130 and ZO1 in transverse sections of HH stage 4 embryos. (A, B) The same microscopic field showing expression of E-cad and/or P-cad (A) and p120-catenin (B). The proteins colocalize at the periphery of cells in the epiblast, primitive streak and mesoderm. (C) Colocalization of E-cad and/or P-cad, GM130 and ZO1in a transverse section of a HH stage 4 embryo. Cells moving from the epiblast to the mesoderm retain E-cad and/or P-cad at their periphery while exhibiting a reorientation of Golgi that is characteristic of the change in cell polarity associated with EMT. (D) Transverse section through a E7.5 mouse embryo at the level of the primitive streak, visualizing E-cad and/or P-cad proteins. (E) Higher magnification of the boxed area in (D), showing persistence of E-cad and/or P-cad proteins on the surface of cells in the mesoderm below and near the primitive streak (arrow). Asterisks are rounded cells in the mesoderm that retain E-cad and/or P-cad protein.</p