Session Chairs: Ke Lan and Wolfgang Hammerschmidt [9:00 AM – 10:30 AM]
Oral Talk #2
Global Changes in the Host B cell During the Pre-latent Phase of EBV Infection
Paulina Mrozek-Gorska1, Alexander Buschle1, Antonio Scialdone2, Thomas Schwarzmayr3, Wolfgang Hammerschmidt1
1Helmholtz Centre Munich Research Unit Gene Vectors, 2Helmholt Centre Munich Institute of Epigenetics and Stem Cells, 3Helmholtz Centre Munich Institute of Human Genetics
In vitro infection of human quiescent B cells with EBV activates them and leads to infinitely proliferating lymphoblastoid B cell lines (LCL). In this process, the virus triggers dramatic changes in the host cell biology to allow its long-term persistence and to guarantee the survival of its host cell in the latent phase. I investigated the very early steps of EBV infection focusing on changes in the host cell biology. Early after infection cells grow in cell volume and massively induce RNA and proteins synthesis. Within the first two days, the cells show stable metabolic activity and do not proliferate. On day three post infection (p.i), the cells initiate DNA replication and cell division followed by a phase of intense proliferation. Starting at day four p.i., the cells show very high metabolic activities characterized by an increased uptake of glucose and enhanced mitochondrial activity. I performed time-resolved RNA-seq experiments to follow the dynamics and kinetics of transcriptome changes upon EBV infection. The analyses of these experiments identified seven different clusters of genes with very specific gene expression patterns in EBV- infected cells compared with uninfected cells. My results demonstrate that the virus governs all important cellular processes including proliferation, cell metabolism, and various epigenetic mechanisms in a strictly time-controlled manner during infection.
The virus delivers its epigenetically naïve genomic DNA to the host cell upon infection, but during latency the viral DNA is extrachromosomaly maintained and organized with nucleosomes including mostly repressive histone marks. I investigated the kinetics of nucleosome assembly on viral DNA as well as the positioning of nucleosomes. Within 24 hours after infection, EBV DNA had acquired nucleosomal structures, which accumulated and became more prevalent until day three p.i.. Nucleosome acquisition did not appear to be random, but pre-defined locations occupied with nucleosomes were found very early p.i..
Oral Talk #3
Single-Cell Analysis of Gammaherpesvirus Infection and Gene Expression
Lauren Oko1, Rachael Kasper2, Abigail Kimbail2, Tim Chang3, Benjamin Alderete3, Linda F. van Dyk1, Eric T. Clambey2
1University of Colorado Denver Anschutz Medical Campus, Department of Microbiology & Immunology, 2University of Colorado Denver Anschutz Medical Campus, Department of Anesthesiology, 3MilliporeSigma
Virus-host interactions are traditionally investigated in pooled cell populations which may conceal cell-to-cell variation. While gammaherpesvirus gene expression is well understood at a population level, little is known about viral gene expression in individual cells. Here, we made use of the PrimeFlowTM RNA assay, a highly sensitive fluorescent in situ hybridization platform, to investigate endogenous viral and host RNA expression and degradation during gammaherpesvirus infection. This technique allowed simultaneous measurement of viral and host RNAs (mRNA and ncRNAs) and proteins in a high- throughput manner, using multiparameter flow cytometry. We applied this technology to explore viral and host gene expression in KSHV, EBV and gammaherpesvirus 68 (γHV68) using multiple in vitro infection systems. These studies identified that viral non-coding RNAs provide a robust and reliable marker for viral infection, and revealed heterogeneous expression in different conditions. Further, by analyzing lytic and latent γHV68 infection, we found marked heterogeneity in gene expression at the single cell level. Whereas lytic infection is characterized by a distribution of cells with varying levels of viral RNAs, host mRNA degradation, and divergent RNA localization, reactivation from latency is characterized by rare cells demonstrating robust gene expression. In total, these data emphasize the heterogeneity of gammaherpesvirus gene expression at the single-cell level.
Oral Talk #4
3D architecture of host-pathogen genomic interactions during EBV reactivation
Stephanie Moquin1,2, Sean Thomas2, Sean Whalen1,2, Samantha Fernandez2, Katherine Pollard1,2, JJ Miranda1,2 1University of California, San Francisco, 2Gladstone Institutes
The episomal genome of the human Epstein-Barr virus (EBV) contacts human chromosomes in 3D space in a non-random manner that changes upon reactivation. We have measured and characterized these host-pathogen interactions using high-throughput chromatin conformation capture (HiC). During latency, EBV localizes to human regions that are regions with characteristics of heterochromatin: gene-poor and AT-rich. Contact points are enriched at the viral latent origin of replication oriP, but perhaps surprisingly, spatial localization appears not dependent on the protein EBNA1. A machine learning algorithm analyzing hundreds of ENCODE datasets identifies a cohort of ~10 proteins on each genome that preferentially occupy host-pathogen contact points. Upon reactivation, the genome-wide set of contacts reorganizes away from heterochromatin and toward euchromatin. We argue that this represents an effort by the virus to surround the episome with human chromatin that corresponds to the desired state of gene expression: quiescent during latency and active during lytic replication. Our current efforts focus on functional and mechanistic understanding of this 3D architecture.
J Virol 2017 The Epstein-Barr virus episome maneuvers between nuclear chromatin compartments during reactivation. (PubMed)
Oral Talk #5
Modulation of Cellular CpG DNA Methylation by Kaposi’s Sarcoma Associated Herpesvirus (Guy Journo)
Journo G, Tushinsky C, Shterngas A, Avital N, Eran Y, Karpuj MV, Frenkel-Morgenstern M, Shamay M.
J Virol. 2018 Jun 13. PMID: 29899086
Kaposi’s sarcoma associated herpesvirus (KSHV, HHV-8) is a gamma herpesvirus associated with several human malignancies. DNA methylation at CpG dinucleotides is an epigenetic mark dysregulated in many cancer types. Several previous studies have analyzed in detail the CpG methylation of the Kaposi’s sarcoma associated herpesvirus (KSHV, HHV-8) episomal genomes, but little is known about the impact of KSHV on the human genome. Our knowledge of cellular CpG methylation in the context of KSHV infection is currently limited to four hyper-methylated human gene promoters. Therefore, we undertook a comprehensive CpG methylation analysis of the human methylome in KSHV-infected cells and KSHV-associated primary effusion lymphoma (PEL). We performed Infinium HumanMethylation 450K and EPIC BeadChip arrays and identified panels of hyper and hypo-methylated cellular promoters in KSHV infected cells. We combined our genome wide methylation analysis with RNA-sequencing (RNA-seq) to add functional outcomes to the virally induced methylation changes. We were able to correlate many downregulated genes with promoter hyper-methylation, and upregulated genes with hypo-methylation. In addition, we show that treating the cells with a de-methylating agent leads to re-expression of these downregulated genes, indicating that indeed DNA methylation plays a role in the repression of these human genes. Comparison between de- novo infection and PEL, suggests that the virus induces initial hyper-methylation followed with a slow increase in genome wide hypo-methylation. This study extends our understanding of the relationship between epigenetic changes induced by KSHV infection and tumorigenesis.
Transforming Naive B Cells, and Supports the Activation of
Viral Genes, but not EBNA2-Induced Cellular Genes
Agnieszka Szymula1,2, Richard D Palermo1, Ian J Groves1, Beth Holder1, Mohammed Ba abdullah1, Amr Bayoumy1, Rob E White1
1Section of Virology, Imperial College, 2Harvard University
PLoS Pathog. 2018 Feb 20;14(2):e1006890.
The Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is the first viral latency-associated protein produced after EBV infection of resting B cells. Its role in B cell transformation is poorly defined, but it has been reported to enhance gene activation by the EBV protein EBNA2 in vitro. Using an EBNA-LP knockout (LPKO) EBV, containing a STOP codon within each repeat unit of IR1, we found that EBNA-LP-mutant EBVs established lymphoblastoid cell lines (LCLs) from adult B cells at reduced efficiency, but not from umbilical cord B cells, which died approximately two weeks after infection. Notably, LPKO LCLs established from adult B cells exhibited an exclusively memory B cell phenotype. Complementation experiments are underway to further explore these observations.
Quantitative PCR analysis of virus gene expression after infection showed reduced transcription of EBNA3, LMP and EBER genes, with only EBNA2 unaffected. Levels of all of these transcripts returned to normal levels as LCLs were established. In contrast, EBNA2-induced cellular genes were activated efficiently by LPKO viruses, sometimes to higher levels than the wild-type virus. RNA-seq analysis 2 days post infection confirms that EBNA2 target genes are induced efficiently in the absence of EBNA-LP. Chromatin immunoprecipitation revealed that EBNA2 and the host transcription factors EBF1 and RBPJ were delayed in their recruitment to all latency promoters tested, whereas these same factors were recruited efficiently to several host genes, which exhibited increased EBNA2 recruitment. We conclude that EBNA-LP does not simply co-operate with EBNA2 in activating gene transcription, but rather facilitates the recruitment of transcription factors to the viral genome, to enable transcription of virus latency genes, and that EBNA-LP is essential for the survival of EBV-infected naïve B cells.
Comparative Study
Thomas Günther1, Shinji Ohno2, Lia Burkhardt1, Jacqueline Fröhlich1, Heiko Adler2, Adam Grundhoff
1Heinrich Pette Institute, Leibniz Institute for Experimental Virology, 2Helmholtz Zentrum München, German Research Center for Environmental Health
We have previously demonstrated that de novo infecting KSHV episomes rapidly and globally acquire H3K27-me3, a histone modification associated with polycomb repressive complexes (PRCs). While the major latency promoter escapes H3K27 tri-methylation, key lytic promoters acquire bivalent chromatin (characterized by co-occupancy of H3K27-me3 and activating H3K4-me3 marks), suggesting that PRC recruitment is an important event in the establishment of latent viral chromatin. Interestingly, polycomb group proteins (PcG) have recently emerged as important latency-promoting factors for diverse viruses, including CMV, HSV-1 and HIV. While this may suggest a broad role for PRCs in restricting viral DNA transcription, the molecular mechanisms mediating PRC recruitment remain poorly understood. To elucidate whether PRC silencing constitutes a general hallmark of herpesvirus latency, we therefore performed a comparative viral epigenome and transcriptome study between KSHV and the related murine gammaherpesvirus 68 (MHV- 68). Surprisingly, we find that latently replicating MHV-68 episome, while acquiring persistent activation-associated histone modifications that pre-mark early lytic genes for rapid reactivation, fail to efficiently attract H3K27-me3 marks. Co-infection experiments and experiments using recombinant viruses demonstrate that this is a virus specific phenotype which is independent of the host cellular environment, or unique functions mediated by latency-associated nuclear antigens of the two viruses. Instead, we present ChIP-seq and live-cell imaging data which suggest that PRC recruitment to KSHV genomes proceeds via the non-canonical pathway, a recently discovered mechanism which controls the epigenetic state of host CpG islands. Based on our results, we propose that some latently infecting viruses have adopted distinct genomic features to specifically exploit common host pathways that repress epigenetically naive DNA, while others have evolved to evade PRC silencing. The implications of our findings for other nuclear DNA viruses in- and outside of the herpesvirus family will be discussed.