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

Alternative promoters drive human cytomegalovirus reactivation from latency

Reactivation from latency requires reinitiation of viral gene expression and culminates in the production of infectious progeny. The major immediate early promoter (MIEP) of human cytomegalovirus (HCMV) drives the expression of crucial lytic cycle transactivators but is silenced during latency in hematopoietic progenitor cells (HPCs). Because the MIEP has poor activity in HPCs, it is unclear how viral transactivators are expressed during reactivation. It has been presumed that viral gene expression is reinitiated via de-repression of the MIEP. We demonstrate that immediate early transcripts arising from reactivation originate predominantly from alternative promoters within the canonical major immediate early locus. Disruption of these intronic promoters results in striking defects in re-expression of viral genes and viral genome replication in the THP-1 latency model. Furthermore, we show that these promoters are necessary for efficient reactivation in primary CD34+ HPCs. Our findings shift the paradigm for HCMV reactivation by demonstrating that promoter switching governs reactivation from viral latency in a context-specific manner

Measurement of high-p_T Single Electrons from Heavy-Flavor Decays in p+p Collisions at sqrt(s) = 200 GeV

The momentum distribution of electrons from decays of heavy flavor (charm and beauty) for midrapidity |y| < 0.35 in p+p collisions at sqrt(s) = 200 GeV has been measured by the PHENIX experiment at the Relativistic Heavy Ion Collider (RHIC) over the transverse momentum range 0.3 < p_T < 9 GeV/c. Two independent methods have been used to determine the heavy flavor yields, and the results are in good agreement with each other. A fixed-order-plus-next-to-leading-log pQCD calculation agrees with the data within the theoretical and experimental uncertainties, with the data/theory ratio of 1.72 +/- 0.02^stat +/- 0.19^sys for 0.3 < p_T < 9 GeV/c. The total charm production cross section at this energy has also been deduced to be sigma_(c c^bar) = 567 +/- 57^stat +/- 224^sys micro barns.Comment: 375 authors from 57 institutions, 6 pages, 3 figures. Submitted to Physical Review Letters. Plain text data tables for the points plotted in figures for this and previous PHENIX publications are (or will be) publicly available at http://www.phenix.bnl.gov/papers.htm

The Protein-Protein Interaction tasks of BioCreative III: classification/ranking of articles and linking bio-ontology concepts to full text

BACKGROUND: Determining usefulness of biomedical text mining systems requires realistic task definition and data selection criteria without artificial constraints, measuring performance aspects that go beyond traditional metrics. The BioCreative III Protein-Protein Interaction (PPI) tasks were motivated by such considerations, trying to address aspects including how the end user would oversee the generated output, for instance by providing ranked results, textual evidence for human interpretation or measuring time savings by using automated systems. Detecting articles describing complex biological events like PPIs was addressed in the Article Classification Task (ACT), where participants were asked to implement tools for detecting PPI-describing abstracts. Therefore the BCIII-ACT corpus was provided, which includes a training, development and test set of over 12,000 PPI relevant and non-relevant PubMed abstracts labeled manually by domain experts and recording also the human classification times. The Interaction Method Task (IMT) went beyond abstracts and required mining for associations between more than 3,500 full text articles and interaction detection method ontology concepts that had been applied to detect the PPIs reported in them.RESULTS:A total of 11 teams participated in at least one of the two PPI tasks (10 in ACT and 8 in the IMT) and a total of 62 persons were involved either as participants or in preparing data sets/evaluating these tasks. Per task, each team was allowed to submit five runs offline and another five online via the BioCreative Meta-Server. From the 52 runs submitted for the ACT, the highest Matthew's Correlation Coefficient (MCC) score measured was 0.55 at an accuracy of 89 and the best AUC iP/R was 68. Most ACT teams explored machine learning methods, some of them also used lexical resources like MeSH terms, PSI-MI concepts or particular lists of verbs and nouns, some integrated NER approaches. For the IMT, a total of 42 runs were evaluated by comparing systems against manually generated annotations done by curators from the BioGRID and MINT databases. The highest AUC iP/R achieved by any run was 53, the best MCC score 0.55. In case of competitive systems with an acceptable recall (above 35) the macro-averaged precision ranged between 50 and 80, with a maximum F-Score of 55. CONCLUSIONS: The results of the ACT task of BioCreative III indicate that classification of large unbalanced article collections reflecting the real class imbalance is still challenging. Nevertheless, text-mining tools that report ranked lists of relevant articles for manual selection can potentially reduce the time needed to identify half of the relevant articles to less than 1/4 of the time when compared to unranked results. Detecting associations between full text articles and interaction detection method PSI-MI terms (IMT) is more difficult than might be anticipated. This is due to the variability of method term mentions, errors resulting from pre-processing of articles provided as PDF files, and the heterogeneity and different granularity of method term concepts encountered in the ontology. However, combining the sophisticated techniques developed by the participants with supporting evidence strings derived from the articles for human interpretation could result in practical modules for biological annotation workflows

Production of He-4 and (4) in Pb-Pb collisions at root(NN)-N-S=2.76 TeV at the LHC

Results on the production of He-4 and (4) nuclei in Pb-Pb collisions at root(NN)-N-S = 2.76 TeV in the rapidity range vertical bar y vertical bar <1, using the ALICE detector, are presented in this paper. The rapidity densities corresponding to 0-10% central events are found to be dN/dy4(He) = (0.8 +/- 0.4 (stat) +/- 0.3 (syst)) x 10(-6) and dN/dy4 = (1.1 +/- 0.4 (stat) +/- 0.2 (syst)) x 10(-6), respectively. This is in agreement with the statistical thermal model expectation assuming the same chemical freeze-out temperature (T-chem = 156 MeV) as for light hadrons. The measured ratio of (4)/He-4 is 1.4 +/- 0.8 (stat) +/- 0.5 (syst). (C) 2018 Published by Elsevier B.V.Peer reviewe

Direct photon elliptic flow in Pb-Pb collisions at root s(NN)=2.76 TeV

The elliptic flow of inclusive and direct photons was measured at mid-rapidity in two centrality classes 0-20% and 20-40% in Pb-Pb collisions at root s(NN) = 2.76 TeV by ALICE. Photons were detected with the highly segmented electromagnetic calorimeter PHOS and via conversions in the detector material with the e(broken vertical bar)e pairs reconstructed in the central tracking system. The results of the two methods were combined and the direct-photon elliptic flow was extracted in the transverse momentum range 0.9 < p(T) < 6.2 GeV/c. A comparison to RHIC data shows a similar magnitude of the measured direct-photon elliptic flow. Hydrodynamic and transport model calculations are systematically lower than the data, but are found to be compatible. (C) 2018 The Author. Published by Elsevier B.V.Peer reviewe

Long and Short Isoforms of the Human Cytomegalovirus UL138 Protein Silence IE Transcription and Promote Latency

The UL133–138 locus present in clinical strains of human cytomegalovirus (HCMV) encodes proteins required for latency and reactivation in CD34 + hematopoietic progenitor cells and virion maturation in endothelial cells. The encoded proteins form multiple homo- and hetero-interactions and localize within secretory membranes. One of these genes, UL136 gene, is expressed as at least five different protein isoforms with overlapping and unique functions. Here we show that another gene from this locus, the UL138 gene, also generates more than one protein isoform. A long form of UL138 (pUL138-L) initiates translation from codon 1, possesses an amino-terminal signal sequence, and is a type one integral membrane protein. Here we identify a short protein isoform (pUL138-S) initiating from codon 16 that displays a subcellular localization similar to that of pUL138-L. Reporter, short-term transcription, and long-term virus production assays revealed that both pUL138-L and pUL138-S are able to suppress major immediate early (IE) gene transcription and the generation of infectious virions in cells in which HCMV latency is studied. The long form appears to be more potent at silencing IE transcription shortly after infection, while the short form seems more potent at restricting progeny virion production at later times, indicating that both isoforms of UL138 likely cooperate to promote HCMV latency. IMPORTANCE Latency allows herpesviruses to persist for the lives of their hosts in the face of effective immune control measures for productively infected cells. Controlling latent reservoirs is an attractive antiviral approach complicated by knowledge deficits for how latently infected cells are established, maintained, and reactivated. This is especially true for betaherpesviruses. The functional consequences of HCMV UL138 protein expression during latency include repression of viral IE1 transcription and suppression of virus replication. Here we show that short and long isoforms of UL138 exist and can themselves support latency but may do so in temporally distinct manners. Understanding the complexity of gene expression and its impact on latency is important for considering potential antivirals targeting latent reservoirs.HHS \ NIH \ NIH Office of the Director (OD) [AI074984, AI079059]; NSF \ NSF Office of the Director (OD)Accepted manuscript posted online 10 August 2016; 6 month embargo.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]

EGFR signaling maintains latency.

<p>CD34⁺ HPCs were infected with WT, <i>UL135</i>_<i>STOP</i>, and <i>UL138</i>_<i>STOP</i> virus at an MOI of 2. (A) At 1, 4, and 8 dpi cells were stained with Alexa Fluor 647-conjugated EGF ligand and BV421-conjugated ms α-CD34 for FACS analysis to measure EGFR surface levels. Bars represent the average of three independent experiments where EGFR surface levels are relative to mock infection. Standard error of the mean is shown. Asterisk indicates significance by Student’s t-test where p<0.05. (B) Pure populations of CD34⁺ HPCs infected with WT, <i>UL135</i>_STOP, or <i>UL138</i>_STOP at an MOI of 2 were isolated by FACS at 24 hpi and maintained in LTBMC with EGFR inhibitor AG1478 at 10 μM. At 10 dpi, viable CD34⁺ HPCs were seeded by limiting dilution onto monolayers of permissive fibroblasts (reactivation). An equivalent number of cells were mechanically disrupted and seeded in parallel to determine the infectious virus present in the culture prior to reactivation (pre-reactivation). Frequency of infectious centers formed pre and post reactivation was determined 14 days later from the number of GFP-positive wells at each dilution using ELDA software. Bars represent the average fold change in drug-treated cells relative to vehicle (DMSO) pre-reactivation control for three independent experiments. Standard error of the mean is shown. (C) WT-infected CD34+ cells treated with PI3K inhibitor LY294002 at 20 μM were analyzed as described for panel B. (B-C) Significance determined by two-way ANOVA with Bonferroni correction for differences between vehicle controls and drug treatments, asterisk indicates p-value<0.05.</p

pUL135 and pUL138 impact phosphorylation of EGFR.

<p>Fibroblasts were infected with WT, <i>UL135</i>_STOP, and <i>UL138</i>_<i>STOP</i> virus at an MOI of 1. At 48 hpi, infected cells were pulsed with 10nM EGF for 15min and then lysed. (A) EGFR was immunoprecipitated with ms α-EGFR and both IP and lysate samples were separated by SDS-PAGE. Blots were stained with rb α-EGFR, rb α-EGFR phosphotyrosine 1068, ms α-phosphotyrosine, and ms α-IE1/2. (B) The quantification of phosphorylation over three experiments is shown. To control for the variation in EGFR levels in different infections, we normalized signals associated with pY1068 or pY to total EGFR. Statistical significance relative to WT was calculated by student t-test (* p-value ≤ 0.05). (C) Serum-starved or fed fibroblasts expressing EGFR_3XFLAG were infected with WT CMV at 20 hours post transduction. Cells were stained with ms α-EGFR, rb α-EGFR pY1068 at 48 hpi. A merge of all three images in shown to the right. (D) Serum-starved, infected fibroblasts were pulsed with Alexa Fluor 647-conjugated EGF ligand on ice, fixed 20 min after a shift to 37C and stained with rb α-EGFR. Cells were imaged by deconvolution microscopy. For C and D, nuclei are stained with DAPI.</p