Thomas Günther

The Epigenetic Landscape of Latent Kaposi Sarcoma-Associated Herpesvirus Genomes

Herpesvirus latency is generally thought to be governed by epigenetic modifications, but the dynamics of viral chromatin at early timepoints of latent infection are poorly understood. Here, we report a comprehensive spatial and temporal analysis of DNA methylation and histone modifications during latent infection with Kaposi Sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi Sarcoma and primary effusion lymphoma (PEL). By use of high resolution tiling microarrays in conjunction with immunoprecipitation of methylated DNA (MeDIP) or modified histones (chromatin IP, ChIP), our study revealed highly distinct landscapes of epigenetic modifications associated with latent KSHV infection in several tumor-derived cell lines as well as de novo infected endothelial cells. We find that KSHV genomes are subject to profound methylation at CpG dinucleotides, leading to the establishment of characteristic global DNA methylation patterns. However, such patterns evolve slowly and thus are unlikely to control early latency. In contrast, we observed that latency-specific histone modification patterns were rapidly established upon a de novo infection. Our analysis furthermore demonstrates that such patterns are not characterized by the absence of activating histone modifications, as H3K9/K14-ac and H3K4-me3 marks were prominently detected at several loci, including the promoter of the lytic cycle transactivator Rta. While these regions were furthermore largely devoid of the constitutive heterochromatin marker H3K9-me3, we observed rapid and widespread deposition of H3K27-me3 across latent KSHV genomes, a bivalent modification which is able to repress transcription in spite of the simultaneous presence of activating marks. Our findings suggest that the modification patterns identified here induce a poised state of repression during viral latency, which can be rapidly reversed once the lytic cycle is induced.

KSHV-Initiated Notch Activation Leads to Membrane-Type-1 Matrix Metalloproteinase-Dependent Lymphatic Endothelial-to-Mesenchymal Transition

Kaposi sarcoma (KS), an angioproliferative disease associated with Kaposi sarcoma herpesvirus (KSHV) infection, harbors a diversity of cell types ranging from endothelial to mesenchymal cells of unclear origin. We developed a three-dimensional cell model for KSHV infection and used it to demonstrate that KSHV induces transcriptional reprogramming of lymphatic endothelial cells to mesenchymal cells via endothelial-to-mesenchymal transition (EndMT). KSHV-induced EndMT was initiated by the viral proteins vFLIP and vGPCR through Notch pathway activation, leading to gain of membrane-type-1 matrix metalloproteinase (MT1-MMP)-dependent invasive properties and concomitant changes in viral gene expression. Mesenchymal markers and MT1-MMP were found codistributed with a KSHV marker in the same cells from primary KS biopsies. Our data explain the heterogeneity of cell types within KS lesions and suggest that KSHV-induced EndMT may contribute to KS development by giving rise to infected, invasive cells while providing the virus a permissive cellular microenvironment for efficient spread.

Kaposi's Sarcoma-Associated Herpesvirus Bacterial Artificial Chromosome Contains a Duplication of a Long Unique Region Fragment Within the Terminal Repeat Region

ABSTRACT Use of the Kaposis sarcoma-associated herpesvirus (KSHV) bacterial artificial chromosome 36 (KSHV-BAC36) genome permits reverse genetics approaches to study KSHV biology. While sequencing the complete KSHV-BAC36 genome, we noted a duplication of a 9-kb fragment of the long unique region in the terminal repeat region. This duplication covers a part of open reading frame (ORF) 19, the complete ORFs 18, 17, 16, K7, K6, and K5, and the putative ORF in the left origin of lytic replication, and it contains the BAC cassette. This observation needs to be kept in mind if viral genes located within the duplicated region are to be mutated in KSHV-BAC36.

Runx1 is essential at two stages of early murine B-cell development

The t(12;21) chromosomal translocation, targeting the gene encoding the RUNX1 transcription factor, is observed in 25% of pediatric acute lymphoblastic leukemia (ALL) and is an initiating event in the disease. To elucidate the mechanism by which RUNX1 disruption initiates leukemogenesis, we investigated its normal role in murine B-cell development. This study revealed 2 critical functions of Runx1: (1) to promote survival and development of progenitors specified to the B-cell lineage, a function that can be substituted by ectopic Bcl2 expression, and (2) to enable the developmental transition through the pre-B stage triggered by the pre-B-cell antigen receptor (pre-BCR). Gene expression analysis and genomewide Runx1 occupancy studies support the hypothesis that Runx1 reinforces the transcription factor network governing early B-cell survival and development and specifically regulates genes encoding members of the Lyn kinase subfamily (key integrators of interleukin-7 and pre-BCR signaling) and the stage-specific transcription factors SpiB and Aiolos (critical downstream effectors of pre-BCR signaling). Interrogation of expression databases of 257 ALL samples demonstrated the specific down-regulation of the SPIB and IKZF3 genes (the latter encoding AIOLOS) in t(12;21) ALL, providing novel insight into the mechanism by which the translocation blocks B-cell development and promotes leukemia.

Activation of the B-cell antigen receptor triggers reactivation of latent Kaposi's Sarcoma-associated Herpesvirus in B-cells

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus and the cause of Kaposis sarcoma, primary effusion lymphoma (PEL) and multicentric Castlemans disease. Latently infected B cells are the main reservoir of this virus in vivo, but the nature of the stimuli that lead to its reactivation in B cells is only partially understood. We established stable BJAB cell lines harboring latent KSHV by cell-free infection with recombinant virus carrying a puromycin resistance marker. Our latently infected B cell lines, termed BrK.219, can be reactivated by triggering the B cell receptor (BCR) with antibodies to surface IgM, a stimulus imitating antigen recognition. Using this B cell model system we studied the mechanisms that mediate the reactivation of KSHV in B cells following the stimulation of the BCR and could identify phosphatidylinositol 3-kinase (PI3K) and X-box binding protein 1 (XBP-1) as proteins that play an important role in the BCR-mediated reactivation of latent KSHV.

Influence of ND10 Components on Epigenetic Determinants of Early KSHV Latency Establishment

We have previously demonstrated that acquisition of intricate patterns of activating (H3K4me3, H3K9/K14ac) and repressive (H3K27me3) histone modifications is a hallmark of KSHV latency establishment. The precise molecular mechanisms that shape the latent histone modification landscape, however, remain unknown. Promyelocytic leukemia nuclear bodies (PML-NB), also called nuclear domain 10 (ND10), have emerged as mediators of innate immune responses that can limit viral gene expression via chromatin based mechanisms. Consequently, although ND10 functions thus far have been almost exclusively investigated in models of productive herpesvirus infection, it has been proposed that they also may contribute to the establishment of viral latency. Here, we report the first systematic study of the role of ND10 during KSHV latency establishment, and link alterations in the subcellular distribution of ND10 components to a temporal analysis of histone modification acquisition and host cell gene expression during the early infection phase. Our study demonstrates that KSHV infection results in a transient interferon response that leads to induction of the ND10 components PML and Sp100, but that repression by ND10 bodies is unlikely to contribute to KSHV latency establishment. Instead, we uncover an unexpected role for soluble Sp100 protein, which is efficiently and permanently relocalized from nucleoplasmic and chromatin-associated fractions into the insoluble matrix. We show that LANA expression is sufficient to induce Sp100 relocalization, likely via mediating SUMOylation of Sp100. Furthermore, we demonstrate that depletion of soluble Sp100 occurs precisely when repressive H3K27me3 marks first accumulate on viral genomes, and that knock-down of Sp100 (but not PML or Daxx) facilitates H3K27me3 acquisition. Collectively, our data support a model in which non-ND10 resident Sp100 acts as a negative regulator of polycomb repressive complex-2 (PRC2) recruitment, and suggest that KSHV may actively escape ND10 silencing mechanisms to promote establishment of latent chromatin.

Rapid Metagenomic Diagnostics for Suspected Outbreak of Severe Pneumonia

To the Editor: Recent outbreaks of severe pneumonia or acute respiratory distress syndrome (ARDS) have attracted much public interest. Given current awareness levels, clinical personnel and health officials must rapidly and adequately respond to suspected outbreaks to prevent public disturbances. We report a case that highlights the potential of next-generation sequencing (NGS) to complement conventional diagnostics in such scenarios.

A Comprehensive Analysis of Replicating Merkel Cell Polyomavirus Genomes Delineates the Viral Transcription Program and Suggests a Role for mcv-miR-M1 in Episomal Persistence

Merkel cell polyomavirus (MCPyV) is considered the etiological agent of Merkel cell carcinoma and persists asymptomatically in the majority of its healthy hosts. Largely due to the lack of appropriate model systems, the mechanisms of viral replication and MCPyV persistence remain poorly understood. Using a semi-permissive replication system, we here report a comprehensive analysis of the role of the MCPyV-encoded microRNA (miRNA) mcv-miR-M1 during short and long-term replication of authentic MCPyV episomes. We demonstrate that cells harboring intact episomes express high levels of the viral miRNA, and that expression of mcv-miR-M1 limits DNA replication. Furthermore, we present RACE, RNA-seq and ChIP-seq studies which allow insight in the viral transcription program and mechanisms of miRNA expression. While our data suggest that mcv-miR-M1 can be expressed from canonical late strand transcripts, we also present evidence for the existence of an independent miRNA promoter that is embedded within early strand coding sequences. We also report that MCPyV genomes can establish episomal persistence in a small number of cells for several months, a time period during which viral DNA as well as LT-Ag and viral miRNA expression can be detected via western blotting, FISH, qPCR and southern blot analyses. Strikingly, despite enhanced replication in short term DNA replication assays, a mutant unable to express the viral miRNA was severely limited in its ability to establish long-term persistence. Our data suggest that MCPyV may have evolved strategies to enter a non- or low level vegetative stage of infection which could aid the virus in establishing and maintaining a lifelong persistence.

Open chromatin structures regulate the efficiencies of pre-RC formation and replication initiation in Epstein-Barr virus

Studies of EBV replication origins demonstrate an excess of pre-replication complexes that are formed at flexible MNase-sensitive sites in the genome.

Recovery of the first full-length genome sequence of a parapoxvirus directly from a clinical sample

We recovered the first full-length poxvirus genome, including the terminal hairpin region, directly from complex clinical material using a combination of second generation short read and third generation nanopore sequencing technologies. The complete viral genome sequence was directly recovered from a skin lesion of a grey seal thereby preventing sequence changes due to in vitro passaging of the virus. Subsequent analysis of the proteins encoded by this virus identified genes specific for skin adaptation and pathogenesis of parapoxviruses. These data warrant the classification of seal parapoxvirus, tentatively designated SePPV, as a new species within the genus Parapoxvirus.