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Journal articleSchmidt N, Hennig T, Serwa RA, et al., 2015,
Remote activation of host cell DNA synthesis in uninfected cells signalled by infected cells in advance of virus transmission.
, Journal of Virology, Vol: 89, Pages: 11107-1115, ISSN: 1098-5514Viruses modulate cellular processes and metabolism in diverse ways but these are almost universally studied in the infected cell itself. Here we study spatial organisation of DNA synthesis during multi-round transmission of herpes simplex virus (HSV) using pulse-labelling with ethynyl-nucleotides and cycloaddition of azide-fluorophores. We report a hitherto unknown and unexpected outcome of virus-host interaction. Consistent with current understanding during single step growth cycle, HSV suppresses host DNA synthesis and promotes virus DNA synthesis in spatially segregated compartments within the cell. In striking contrast, during progressive rounds of infection initiated at a single cell, we observe that infection induces a clear and pronounced stimulation of cellular DNA replication in remote uninfected cells. This induced DNA synthesis was observed in hundreds of uninfected cells at the extended border, outside the perimeter of the progressing infection. Moreover using pulse chase analysis we show that this activation is maintained, resulting in a propagating wave of host DNA synthesis continually in advance of infection. As the virus reaches and infects these activated cells, host DNA synthesis was then shut off and replaced with virus DNA synthesis. Using non-propagating viruses or conditioned medium we demonstrate a paracrine effector of uninfected cell DNA synthesis in remote cells continually in advance of infection. These findings have significant implications, likely with broad applicability, for our understanding of the ways virus infection manipulates cell processes not only in the infected cell itself but also now in remote uninfected cells, as well as for mechanisms governing host DNA synthesis. IMPORTANCE: We show that during infection initiated by a single particle with progressive cell-cell virus transmission (i.e., the normal situation), HSV induces host DNA synthesis in uninfected cells, mediated by a virus induced paracrine effector. The field ha
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Journal articleBarbosa S, Carreira S, Bailey D, et al., 2015,
Phosphorylation and SCF-mediated degradation regulate CREB-H transcription of metabolic targets.
, Molecular Biology of the Cell, Vol: 26, Pages: 2939-2954, ISSN: 1939-4586CREB‑H, an endoplasmic reticulum-anchored transcription factor, plays a key role in regulating secretion and in metabolic and inflammatory pathways, but how its activity is modulated remains unclear. We examined processing of the nuclear active form and identified a motif around S87-S90 with homology to DSG-type phosphodegrons. We show that this region is subject to multiple phosphorylations, which regulate CREB-H stability by targeting it to the SCF(Fbw1a) E3 ubiquitin ligase. Data from phosphatase treatment, use of phosophospecific antibody, and substitution of serine residues demonstrate phosphorylation of candidate serines in the region, with the core S87/S90 motif representing a critical determinant promoting proteasome-mediated degradation. Candidate kinases CKII and GSK-3b phosphorylate CREB-H in vitro with specificities for different serines. Prior phosphorylation with GSK-3 at one or more of the adjacent serines substantially increases S87/S90-dependent phosphorylation by CKII. In vivo expression of a dominant-negative Cul1 enhances steady-state levels of CREB‑H, an effect augmented by Fbw1a. CREB-H directly interacts with Fbw1a in a phosphorylation-dependent manner. Finally, mutations within the phosphodegron, when incorporated into the full-length protein, result in increased levels of constitutively cleaved nuclear protein and increased transcription and secretion of a key endogenous target gene, apolipoprotein A IV.
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Journal articleSerwa R, Krause E, Abaitua F, et al., 2015,
Systems analysis of protein fatty acylation in herpes simplex virus infected cells using chemical proteomics.
, Chemistry & Biology, Vol: 22, Pages: 1008-1017, ISSN: 1074-5521Protein fatty acylation regulates diverse aspects of cellular function and organization and plays a key role in host immune responses to infection. Acylation also modulates the function and localization of virus-encoded proteins. Here, we employ chemical proteomics tools, bio-orthogonal probes, and capture reagents to study myristoylation and palmitoylation during infection with herpes simplex virus (HSV). Using in-gel fluorescence imaging and quantitative mass spectrometry, we demonstrate a generalized reduction in myristoylation of host proteins, whereas palmitoylation of host proteins, including regulators of interferon and tetraspanin family proteins, was selectively repressed. Furthermore, we found that a significant fraction of the viral proteome undergoes palmitoylation; we identified a number of virus membrane glycoproteins, structural proteins, and kinases. Taken together, our results provide broad oversight of protein acylation during HSV infection, a roadmap for similar analysis in other systems, and a resource with which to pursue specific analysis of systems and functions.
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Journal articleElderfield RA, Parker L, Stilwell P, et al., 2015,
Ferret airway epithelial cell cultures support efficient replication of influenza B virus but not mumps virus
, JOURNAL OF GENERAL VIROLOGY, Vol: 96, Pages: 2092-2098, ISSN: 0022-1317- Author Web Link
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- Citations: 5
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Journal articleBrady G, Haas DA, Farrell PJ, et al., 2015,
Poxvirus Protein MC132 from Molluscum Contagiosum Virus Inhibits NF-kappa B Activation by Targeting p65 for Degradation
, JOURNAL OF VIROLOGY, Vol: 89, Pages: 8406-8415, ISSN: 0022-538X -
Journal articleBazot Q, Paschos K, Skalska L, et al., 2015,
Epstein-Barr virus proteins EBNA3A and EBNA3C together induce expression of the oncogenic microRNA cluster miR-221/miR-222 and ablate expression of its target p57KIP2.
, PLOS Pathogens, Vol: 11, ISSN: 1553-7366We show that two host-encoded primary RNAs (pri-miRs) and the corresponding microRNA (miR) clusters - widely reported to have cell transformation-associated activity - are regulated by EBNA3A and EBNA3C. Utilising a variety of EBV-transformed lymphoblastoid cell lines (LCLs) carrying knockout-, revertant- or conditional-EBV recombinants, it was possible to demonstrate unambiguously that EBNA3A and EBNA3C are both required for transactivation of the oncogenic miR-221/miR-222 cluster that is expressed at high levels in multiple human tumours - including lymphoma/leukemia. ChIP, ChIP-seq, and chromosome conformation capture analyses indicate that this activation results from direct targeting of both EBV proteins to chromatin at the miR-221/miR-222 genomic locus and activation via a long-range interaction between enhancer elements and the transcription start site of a long non-coding pri-miR located 28kb upstream of the miR sequences. Reduced levels of miR-221/miR-222 produced by inactivation or deletion of EBNA3A or EBNA3C resulted in increased expression of the cyclin-dependent kinase inhibitor p57KIP2, a well-established target of miR-221/miR-222. MiR blocking experiments confirmed that miR-221/miR-222 target p57KIP2 expression in LCLs. In contrast, EBNA3A and EBNA3C are necessary to silence the tumour suppressor cluster miR-143/miR-145, but here ChIP-seq suggests that repression is probably indirect. This miR cluster is frequently down-regulated or deleted in human cancer, however, the targets in B cells are unknown. Together these data indicate that EBNA3A and EBNA3C contribute to B cell transformation by inhibiting multiple tumour suppressor proteins, not only by direct repression of protein-encoding genes, but also by the manipulation of host long non-coding pri-miRs and miRs.
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Journal articleHennig T, O'Hare P, 2015,
Viruses and the nuclear envelope
, CURRENT OPINION IN CELL BIOLOGY, Vol: 34, Pages: 113-121, ISSN: 0955-0674- Author Web Link
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- Citations: 19
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Journal articleBamford A, Crowe BHA, Hacohen Y, et al., 2015,
Pediatric Herpes Simplex Virus Encephalitis Complicated by N-Methyl-D-aspartate Receptor Antibody Encephalitis
, JOURNAL OF THE PEDIATRIC INFECTIOUS DISEASES SOCIETY, Vol: 4, Pages: E17-E21, ISSN: 2048-7193- Author Web Link
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- Citations: 21
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Journal articlePalser AL, Grayson NE, White RE, et al., 2015,
Genome Diversity of Epstein-Barr Virus from Multiple Tumor Types and Normal Infection
, Journal of Virology, Vol: 89, Pages: 5222-5237, ISSN: 1098-5514Epstein-Barr virus (EBV) infects most of the world’s population and is causally associated with several human cancers, but littleis known about how EBV genetic variation might influence infection or EBV-associated disease. There are currently no publishedwild-type EBV genome sequences from a healthy individual and very few genomes from EBV-associated diseases. We havesequenced 71 geographically distinct EBV strains from cell lines, multiple types of primary tumor, and blood samples and thefirst EBV genome from the saliva of a healthy carrier. We show that the established genome map of EBV accurately represents allstrains sequenced, but novel deletions are present in a few isolates. We have increased the number of type 2 EBV genomes sequencedfrom one to 12 and establish that the type 1/type 2 classification is a major feature of EBV genome variation, definedalmost exclusively by variation of EBNA2 and EBNA3 genes, but geographic variation is also present. Single nucleotide polymorphism(SNP) density varies substantially across all known open reading frames and is highest in latency-associated genes. SomeT-cell epitope sequences in EBNA3 genes show extensive variation across strains, and we identify codons under positive selection,both important considerations for the development of vaccines and T-cell therapy. We also provide new evidence for recombinationbetween strains, which provides a further mechanism for the generation of diversity. Our results provide the firstglobal view of EBV sequence variation and demonstrate an effective method for sequencing large numbers of genomes to furtherunderstand the genetics of EBV infection.
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Journal articleSerrao E, Ballandras-Colas A, Cherepanov P, et al., 2015,
Key determinants of target DNA recognition by retroviral intasomes
, Retrovirology, Vol: 12, ISSN: 1742-4690Background: Retroviral integration favors weakly conserved palindrome sequences at the sites of viral DNA joiningand generates a short (4–6 bp) duplication of host DNA flanking the provirus. We previously determined two keyparameters that underlie the target DNA preference for prototype foamy virus (PFV) and human immunodeficiencyvirus type 1 (HIV-1) integration: flexible pyrimidine (Y)/purine (R) dinucleotide steps at the centers of the integrationsites, and base contacts with specific integrase residues, such as Ala188 in PFV integrase and Ser119 in HIV-1 integrase.Here we examined the dinucleotide preference profiles of a range of retroviruses and correlated these findings withrespect to length of target site duplication (TSD).Results: Integration datasets covering six viral genera and the three lengths of TSD were accessed from the literatureor generated in this work. All viruses exhibited significant enrichments of flexible YR and/or selection against rigid RYdinucleotide steps at the centers of integration sites, and the magnitude of this enrichment inversely correlated withTSD length. The DNA sequence environments of in vivo-generated HIV-1 and PFV sites were consistent with integrationinto nucleosomes, however, the local sequence preferences were largely independent of target DNA chromatinization.Integration sites derived from cells infected with the gammaretrovirus reticuloendotheliosis virus strain A (Rev-A),which yields a 5 bp TSD, revealed the targeting of global chromatin features most similar to those of Moloneymurine leukemia virus, which yields a 4 bp duplication. In vitro assays revealed that Rev-A integrase interacts withand is catalytically stimulated by cellular bromodomain containing 4 protein.Conclusions: Retroviral integrases have likely evolved to bend target DNA to fit scissile phosphodiester bondsinto two active sites for integration, and viruses that cut target DNA with a 6 bp stagger may not need to bendDNA as sharply as viruses tha
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