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Lytic herpes simplex virus 1 (HSV-1) infection triggers disruption of transcription termination (DoTT) of most cellular genes, resulting in extensive intergenic transcription. Similarly, cellular stress responses lead to gene-specific transcription downstream of genes (DoG). In this study, we performed a detailed comparison of DoTT/DoG transcription between HSV-1 infection, salt and heat stress in primary human fibroblasts using 4sU-seq and ATAC-seq. Although DoTT at late times of HSV-1 infection was substantially more prominent than DoG transcription in salt and heat stress, poly(A) read-through due to DoTT/DoG transcription and affected genes were significantly correlated between all three conditions, in particular at earlier times of infection. We speculate that HSV-1 either directly usurps a cellular stress response or disrupts the transcription termination machinery in other ways but with similar consequences. In contrast to previous reports, we found that inhibition of Ca2+ signaling by BAPTA-AM did not specifically inhibit DoG transcription but globally impaired transcription. Most importantly, HSV-1-induced DoTT, but not stress-induced DoG transcription, was accompanied by a strong increase in open chromatin downstream of the affected poly(A) sites. In its extent and kinetics, downstream open chromatin essentially matched the poly(A) read-through transcription. We show that this does not cause but rather requires DoTT as well as high levels of transcription into the genomic regions downstream of genes. This raises intriguing new questions regarding the role of histone repositioning in the wake of RNA Polymerase II passage downstream of impaired poly(A) site recognition.

Herpes simplex virus 1 (HSV-1) is an important human pathogen and a paradigm for virus-induced host shut-off. Here we show that global changes in transcription and RNA processing and their impact on translation can be analysed in a single experimental setting by applying 4sU-tagging of newly transcribed RNA and ribosome profiling to lytic HSV-1 infection. Unexpectedly, we find that HSV-1 triggers the disruption of transcription termination of cellular, but not viral, genes. This results in extensive transcription for tens of thousands of nucleotides beyond poly(A) sites and into downstream genes, leading to novel intergenic splicing between exons of neighbouring cellular genes. As a consequence, hundreds of cellular genes seem to be transcriptionally induced but are not translated. In contrast to previous reports, we show that HSV-1 does not inhibit co-transcriptional splicing. Our approach thus substantially advances our understanding of HSV-1 biology and establishes HSV-1 as a model system for studying transcription termination. Herpes simplex virus 1 (HSV-1) efficiently shuts down host gene expression in infected cells. Here Rutkowski et al . analyse the genome-wide changes in transcription and translation in infected cells, and show that HSV-1 triggers an extensive disruption of transcription termination of cellular genes.

The genomes of both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) were first sequenced over 20 years ago. Similar to HCMV, the MCMV genome had initially been proposed to harbor ≈170 open reading frames (ORFs). More recently, omics approaches revealed HCMV gene expression to be substantially more complex comprising several hundred viral ORFs. Here, we provide a state-of-the art reannotation of lytic MCMV gene expression based on integrative analysis of a large set of omics data. Our data reveal 365 viral transcription start sites (TiSS) that give rise to 380 and 454 viral transcripts and ORFs, respectively. The latter include >200 small ORFs, some of which represented the most highly expressed viral gene products. By combining TiSS profiling with metabolic RNA labelling and chemical nucleotide conversion sequencing (dSLAM-seq), we provide a detailed picture of the expression kinetics of viral transcription. This not only resulted in the identification of a novel MCMV immediate early transcript encoding the m166.5 ORF, which we termed ie4 , but also revealed a group of well-expressed viral transcripts that are induced later than canonical true late genes and contain an initiator element (Inr) but no TATA- or TATT-box in their core promoters. We show that viral upstream ORFs (uORFs) tune gene expression of longer viral ORFs expressed in cis at translational level. Finally, we identify a truncated isoform of the viral NK-cell immune evasin m145 arising from a viral TiSS downstream of the canonical m145 mRNA. Despite being ≈5-fold more abundantly expressed than the canonical m145 protein it was not required for downregulating the NK cell ligand, MULT-I. In summary, our work will pave the way for future mechanistic studies on previously unknown cytomegalovirus gene products in an important virus animal model.

5T4 oncofetal molecules are highly expressed during development and upregulated in cancer while showing only low levels in some adult tissues. Upregulation of 5T4 expression is a marker of loss of pluripotency in the early differentiation of embryonic stem (ES) cells and forms an integrated component of an epithelial-mesenchymal transition, a process important during embryonic development and metastatic spread of epithelial tumors. Investigation of the transcriptional changes in early ES differentiation showed upregulation of CXCL12 and down-regulation of a cell surface protease, CD26, which cleaves this chemokine. CXCL12 binds to the widely expressed CXCR4 and regulates key aspects of development, stem cell motility and tumour metastasis to tissues with high levels of CXCL12. We show that the 5T4 glycoprotein is required for optimal functional cell surface expression of the chemokine receptor CXCR4 and CXCL12 mediated chemotaxis in differentiating murine embryonic stem cells and embryo fibroblasts (MEF). Cell surface expression of 5T4 and CXCR4 molecules is co-localized in differentiating ES cells and MEF. By contrast, differentiating ES and MEF derived from 5T4 knockout (KO) mice show only intracellular CXCR4 expression but infection with adenovirus encoding mouse 5T4 restores CXCL12 chemotaxis and surface co-localization with 5T4 molecules. A series of chimeric constructs with interchanged domains of 5T4 and the glycoprotein CD44 were used to map the 5T4 sequences relevant for CXCR4 membrane expression and function in 5T4KO MEF. These data identified the 5T4 transmembrane domain as sufficient and necessary to enable CXCR4 cell surface expression and chemotaxis. Furthermore, some monoclonal antibodies against m5T4 can inhibit CXCL12 chemotaxis of differentiating ES cells and MEF which is not mediated by simple antigenic modulation. Collectively, these data support a molecular interaction of 5T4 and CXCR4 occurring at the cell surface which directly facilitates the biological response to CXCL12. The regulation of CXCR4 surface expression by 5T4 molecules is a novel means to control responses to the chemokine CXCL12 for example during embryogenesis but can also be selected to advantage the spread of a 5T4 positive tumor from its primary site.

Liquid biopsies offer the potential to monitor cancer response and resistance to therapeutics in near real-time. However, the plasma cell free DNA (cfDNA) level can be low and the fraction of circulating tumour DNA (ctDNA) bearing a mutation – lower still. Detection of tumour-derived mutations in ctDNA is thus challenging and requires highly sensitive and specific assays. Droplet digital PCR (ddPCR) is a technique that enables exquisitely sensitive detection and quantification of DNA/RNA markers from very limiting clinical samples, including plasma. The Bio-Rad QX200 ddPCR system provides absolute quantitation of target DNA molecules using fluorescent dual-labelled probes. Critical to accurate sample analysis are validated assays that are highly specific, reproducible, and with known performance characteristics, especially with respect to false positives. We present a systematic approach to the development and optimisation of singleplex and multiplex ddPCR assays for the detection of point mutations with a focus on ensuring extremely low false positives whilst retaining high sensitivity. We also present a refined method to determine cfDNA extraction efficiency allowing for more accurate extrapolation of mutational levels in source samples. We have applied these approaches to successfully analyse many ctDNA samples from multiple clinical studies and generated exploratory data of high quality.

The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution. Here, using computational integration of multi-omics data, the authors provide a detailed transcriptome and translatome of herpes simplex virus 1 (HSV-1), including previously unidentified ORFs and N-terminal extensions. The study also provides a HSV-1 genome browser and should be a valuable resource for further research.

Bone marrow-derived cells have been postulated as a source of multipotent mesenchymal stem cells (MSC). However, the whole fraction of MSC remains heterogeneous and the expansion of primitive subset of these cells is still not well established. Here, we optimized the protocol for propagating the low-adherent subfraction of MSC which results in long-term expansion of population characterized by CD45-CD14+CD34+ phenotype along with expression of common MSC markers. We established that the expanded MSC are capable of differentiating into endothelial cells highly expressing angiogenic markers and exhibiting functional properties of endothelium. Moreover, we found these cells to be multipotent and capable of giving rise into cells from neuronal lineages. Interestingly, the expanded MSC form characteristic cellular spheres in vitro indicating primitive features of these cells. In sum, we isolated the novel multipotent subpopulation of CD45-CD14+CD34+ bone marrow-derived cells that could be maintained in long-term culture without losing this potential.

Effective vaccination against tumour-associated antigens (TAA) such as the 5T4 oncofoetal glycoprotein may be limited by the nature of the T cell repertoire and the influence of immunomodulatory factors in particular T regulatory cells (Treg). Here, we identified mouse 5T4-specific T cell epitopes using a 5T4 knock out (5T4KO) mouse and evaluated corresponding wild-type (WT) responses as a model to refine and improve immunogenicity. We have shown that 5T4KO mice vaccinated by replication defective adenovirus encoding mouse 5T4 (Adm5T4) generate potent 5T4-specific IFN-γ CD8 and CD4 T cell responses which mediate significant protection against 5T4 positive tumour challenge. 5T4KO CD8 but not CD4 primed T cells also produced IL-17. By contrast, Adm5T4-immunized WT mice showed no tumour protection consistent with only low avidity CD8 IFN-γ, no IL-17 T cell responses and no detectable CD4 T cell effectors producing IFN-γ or IL-17. Treatment with anti-folate receptor 4 (FR4) antibody significantly reduced the frequency of Tregs in WT mice and enhanced 5T4-specific IFN-γ but reduced IL-10 T cell responses but did not reveal IL-17-producing effectors. This altered balance of effectors by treatment with FR4 antibody after Adm5T4 vaccination provided modest protection against autologous B16m5T4 melanoma challenge. The efficacy of 5T4 and some other TAA vaccines may be limited by the combination of TAA-specific T regs, the deletion and/or alternative differentiation of CD4 T cells as well as the absence of distinct subsets of CD8 T cells.

Table of contents Oral presentations Session 1: Entry & uncoating O1 Host cell polo-like kinases (PLKs) promote early prototype foamy virus (PFV) replication Irena Zurnic, Sylvia Hütter, Ute Lehmann, Nicole Stanke, Juliane Reh, Tobias Kern, Fabian Lindel, Gesche Gerresheim, Martin Hamann, Erik Müllers, Paul Lesbats, Peter Cherepanov, Erik Serrao, Alan Engelman, Dirk Lindemann O2 A novel entry/uncoating assay reveals the presence of at least two species of viral capsids during synchronized HIV-1 infection Claire Da Silva Santos, Kevin Tartour, Andrea Cimarelli O3 Dynamics of nuclear envelope association and nuclear import of HIV-1 complexes Rya Burdick, Jianbo Chen, Jaya Sastri, Wei-Shau Hu, Vinay Pathak O4 Human papillomavirus protein E4 potently enhances the susceptibility to HIV infection Oliver T. Keppler Session 2: Reverse transcription & integration O5 Structure and function of HIV-1 integrase post translational modifications Karine Pradeau, Sylvia Eiler, Nicolas Levy, Sarah Lennon, Sarah Cianferani, Stéphane Emiliani, Marc Ruff O6 Regulation of retroviral integration by RNA polymerase II associated factors and chromatin structure Vincent Parissi Session 3: Transcription and latency O7 A novel single-cell analysis pipeline to identify specific biomarkers of HIV permissiveness Sylvie Rato, Antonio Rausell, Miguel Munoz, Amalio Telenti, Angela Ciuffi O8 A capsid-dependent integration program linking T cell activation to HIV-1 gene expression Alexander Zhyvoloup, Anat Melamed, Ian Anderson, Delphine Planas, Janos Kriston-Vizi, Robin Ketteler, Chen-Hsuin Lee, Andy Merritt, Petronela Ancuta, Charles Bangham, Ariberto Fassati O9 Characterisation of new RNA polymerase III and RNA polymerase II transcriptional promoters in the Bovine Leukemia Virus genome Anthony Rodari, Benoit Van Driessche, Mathilde Galais, Nadége Delacourt, Sylvain Fauquenoy, Caroline Vanhulle, Anna Kula, Arsène Burny, Olivier Rohr, Carine Van Lint O10 Tissue-specific dendritic cells differentially modulate latent HIV-1 reservoirs Thijs van Montfort, Renee van der Sluis, Dave Speijer, Ben Berkhout Session 4: RNA trafficking & packaging O11 A novel cis -acting element affecting HIV replication Bo Meng, Andrzej Rutkowski, Neil Berry, Lars Dölken, Andrew Lever O12 Tolerance of HIV’s late gene expression towards stepwise codon adaptation Thomas Schuster, Benedikt Asbach, Ralf Wagner Session 5: Assembly & release O13 Importance of the tax-inducible actin-bundling protein fascin for transmission of human T cell leukemia virus Type 1 (HTLV-1) Christine Gross, Veit Wiesmann, Martina Kalmer, Thomas Wittenberg, Jan Gettemans, Andrea K. Thoma-Kress O14 Lentiviral nef proteins antagonize TIM-mediated inhibition of viral release Minghua Li, Eric O. Freed, Shan-Lu Liu Session 6: Pathogenesis & evolution O15 SEVI and semen prolong the half-life of HIV-1 Janis Müller, Jan Münch O16 CD169 + macrophages mediate retrovirus trans -infection of permissive lymphocytes to establish infection in vivo Xaver Sewald, Pradeep Uchil, Mark Ladinsky, Jagadish Beloor, Ruoxi Pi, Christin Herrmann, Nasim Motamedi, Thomas Murooka, Michael Brehm, Dale Greiner, Thorsten Mempel, Pamela Bjorkman, Priti Kumar, Walther Mothes O17 Efficient replication of a vpu containing SIVagm construct in African Green Monkeys requires an HIV-1 nef gene Simone Joas, Erica Parrish, Clement Wesley Gnanadurai, Edina Lump, Christina M. Stürzel, Nicholas F. Parrish, Ulrike Sauermann, Katharina Töpfer, Tina Schultheiss, Steven Bosinger, Guido Silvestri, Cristian Apetrei, Nicholas Huot, Michaela Müller-Trutwin, Daniel Sauter, Beatrice H. Hahn, Christiane Stahl-Hennig, Frank Kirchhoff O18 Reprogramming initiates mobilization of endogenous mutagenic LINE-1, Alu and SVA retrotransposons in human induced pluripotent stem cells with consequences for host gene expression Gerald Schumann, Sabine Jung-Klawitter, Nina V. Fuchs, Kyle R. Upton, Martin Muñoz-Lopez, Ruchi Shukla, Jichang Wang, Marta Garcia-Canadas, Cesar Lopez-Ruiz, Daniel J. Gerhardt, Attila Sebe, Ivana Grabundzija, Patricia Gerdes, Sylvia Merkert, Andres Pulgarin, Anja Bock, Ulrike Held, Anett Witthuhn, Alexandra Haase, Ernst J. Wolvetang, Ulrich Martin, Zoltán Ivics, Zsuzsanna Izsvák, J. Garcia-Perez, Geoffrey J. Faulkner O19 NF-κB activation induces expression of human endogenous retrovirus and particle production Tara Hurst, Aris Katzourakis, Gkikas Magiorkinis Session 7a and b: Innate sensing & intrinsic immunity O20 Identification of the phosphatase acting on T592 in SAMHD1 during M/G 1 transition Kerstin Schott, Rita Derua, Janna Seifried, Andreas Reuter, Heike Schmitz, Christiane Tondera, Alberto Brandariz-Nuñez, Felipe Diaz-Griffero, Veerle Janssens, Renate König O21 Vpx overcomes a SAMHD1-independent block to HIV reverse transcription that is specific to resting CD4 T cells Hanna-Mari Baldauf, Lena Stegmann, Sarah-Marie Schwarz, Maud Trotard, Margarethe Martin, Gina Lenzi, Manja Burggraf, Xiaoyu Pan, Oliver I. Fregoso, Efrem S. Lim, Libin Abraham, Elina Erikson, Laura Nguyen, Ina Ambiel, Frank Rutsch, Renate König, Baek Kim, Michael Emerman, Oliver T. Fackler, Oliver T. Keppler O22 The role of SAMHD1 in antiviral restriction and immune sensing in the mouse Sabine Wittmann, Rayk Behrendt, Bianca Volkmann, Kristin Eissmann, Thomas Gramberg O23 T cells expressing reduced restriction factors are preferentially infected in therapy naïve HIV-1 patients Sebastian Bolduan, Herwig Koppensteiner, Stefanie Regensburg, Ruth Brack-Werner, Rika Draenert, Michael Schindler O24 cGAS-mediated innate immunity spreads through HIV-1 env-induced membrane fusion sites from infected to uninfected primary HIV-1 target cells Aurélie Ducroux, Shuting Xu, Aparna Ponnurangam, Sergej Franz, Angelina Malassa, Ellen Ewald, Christine Goffinet O25 Perturbation of innate RNA and DNA sensing by human T cell leukemia virus type 1 oncoproteins Sin-Yee Fung, Ching-Ping Chan, Chun-Kit Yuen, Kin-Hang Kok, Chin-Ping Chan, Dong-Yan Jin O26 Induction and anti-viral activity of Interferon α subtypes in HIV-1 infection Ulf Dittmer O27 Vpu-mediated counteraction of tetherin is a major determinant of HIV-1 interferon resistance Dorota Kmiec, Shilpa Iyer, Christina Stürzel, Daniel Sauter, Beatrice Hahn, Frank Kirchhoff O28 DNA repair protein Rad18 restricts HIV-1 and LINE-1 life cycle Yasuo Ariumi, Mariko Yasuda-Inoue, Koudai Kawano, Satoshi Tateishi, Priscilla Turelli O29 Natural mutations in IFITM3 allow escape from post-translational regulation and toggle antiviral specificity Alex Compton, Nicolas Roy, Françoise Porrot, Anne Billet, Nicoletta Casartelli, Jacob Yount, Chen Liang, Oliver Schwartz Session 8: Adaptive immunity & immune evasion O30 Observing evolution in HIV-1 infection: phylogenetics and mutant selection windows to infer the influence of the autologous antibody response on the viral quasispecies Carsten Magnus, Lucia Reh, Penny Moore, Therese Uhr, Jacqueline Weber, Lynn Morris, Alexandra Trkola O31 Dose and subtype specific analyses of the anti-HIV effects of IFN-alpha family members Rashel V. Grindberg, Erika Schlaepfer, Gideon Schreiber, Viviana Simon, Roberto F. Speck Session 9: Novel antiviral strategies O32 LEDGIN-mediated inhibition of the integrase-LEDGF/p75 interaction reduces reactivation of residual latent HIV Zeger Debyser, Lenard Vranckx, Jonas Demeulemeester, Suha Saleh, Eric Verdin, Anna Cereseto, Frauke Christ, Rik Gijsbers O33 NKG2D-mediated clearance of reactivated viral reservoirs by natural killer cells O34 Inhibition of HIV reactivation in brain cells by AAV-mediated delivery of CRISPR/Cas9 O35 CRISPR-Cas9 as antiviral: potent HIV-1 inhibition, but rapid virus escape and the subsequent design of escape-proof antiviral strategies Ben Berkhout, Gang Wang, Na Zhao, Atze T. Das Session 10: Recent advances in HIV vaccine development O36 Priming with a potent HIV-1 DNA vaccine frames the quality of T cell and antibody responses prior to a poxvirus and protein boost Benedikt Asbach, Josef Köstler, Beatriz Perdiguero, Mariano Esteban, Bertram L. Jacobs, David C. Montefiori, Celia C. LaBranche, Nicole L. Yates, Georgia D. Tomaras, Guido Ferrari, Kathryn E. Foulds, Mario Roederer, Gary Landucci, Donald N. Forthal, Michael S. Seaman, Natalie Hawkins, Steven G. Self, Sanjay Phogat, James Tartaglia, Susan W. Barnett, Brian Burke, Anthony D. Cristillo, Song Ding, Jonathan L. Heeney, Giuseppe Pantaleo, Ralf Wagner O37 Passive immunisation with a neutralising antibody against HIV-1 Env prevents infection of the first cells in a mucosal challenge rhesus monkey model Christiane Stahl-Hennig, Viktoria Stab, Armin Ensser, Ulrike Sauermann, Bettina Tippler, Dennis Burton, Matthias Tenbusch, Klaus Überla O38 HIV antibody Fc-glycoforms drive B cell affinity maturation Galit Alter, Giuseppe Lofano, Anne-Sophie Dugast, Viraj Kulkarni, Todd Suscovich Poster presentations Topic 1: Entry & uncoating P1 Dynein light chain is required for murine leukemia virus infection Tatiana Opazo, Felipe Barraza, Diego Herrera, Andrea Garces, Tomas Schwenke, Diego Tapia, Jorge Cancino, Gloria Arriagada P2 Peptide paratope mimics of the broadly neutralising HIV-1 antibody b12 Christina Haußner, Dominik Damm, Anette Rohrhofer, Barbara Schmidt, Jutta Eichler P3 Investigating cellular pathways involved in the transmission of HIV-1 between dendritic cells and T cells using RNAi screening techniques Rebecca Midgley, James Wheeldon, Vincent Piguet P4 Co-receptor tropism in HIV-1, HIV-2 monotypic and dual infections Priyanka Khopkar, Megha Rohamare, Smita Kulkarni P5 Characterisation of the role of CIB1 and CIB2 as HIV-1 helper factors Ana Godinho-Santos, Allan Hance, Joao Goncalves, Fabrizio Mammano P6 Buffering deleterious polymorphisms in the highly constrained C2 region of HIV-1 envelope by the flexible V3 domain Romain Gasser, Meriem Hamoudi, Martina Pellicciotta, Zhicheng Zhou, Clara Visdeloup, Philippe Colin, Martine Braibant, Bernard Lagane, Matteo Negroni P7 Entry inhibition of HERV-K(HML-2)

Although the overall prognosis in childhood acute lymphoblastic leukaemia (ALL) is good, outcome after relapse is poor. Recurrence is frequently characterised by the occurrence of disease at extramedullary sites such as the central nervous system and testes. Subpopulations of blasts able to migrate to such areas may have a survival advantage and give rise to disease recurrence. Gene expression profiling of 85 diagnostic pre-B-ALL bone marrow samples revealed higher 5T4 oncofoetal antigen transcript levels in cytogenetic high-risk subgroups of patients (p < 0.001). Flow cytometric analysis determined that bone marrow from relapse patients have a significantly higher percentage of 5T4 positive leukemic blasts than healthy donors (p = 0.005). The high-risk Sup-B15 pre-B-ALL line showed heterogeneity in 5T4 expression, and the derived, 5T4+ (Sup5T4) and 5T4− (Sup) subline cells, displayed differential spread to the omentum and ovaries following intraperitoneal inoculation of immunocompromised mice. Consistent with this, Sup5T4 compared to Sup cells show increased invasion in vitro concordant with increased LFA-1 and VLA-4 integrin expression, adhesion to extracellular matrix and secretion of matrix metalloproteases (MMP-2/-9). We also show that 5T4 positive Sup-B15 cells are susceptible to 5T4 specific superantigen antibody-dependent cellular toxicity providing support for targeted immunotherapy in high risk pre-B-ALL.