Biology subjects

Grundhoff, A.

Publications and source records attributed to Grundhoff, A..

24 records · Page 2Linked to original sources

bioRxiv2020

High-resolution analysis of Merkel Cell Polyomavirus in Merkel Cell Carcinoma reveals distinct integration patterns and suggests NHEJ and MMBIR as underlying mechanisms

Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas (MCC). MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes. Which molecular mechanisms promote viral integration, if distinct integration patterns exist, and if integration occurs preferentially at loci with specific chromatin states is unknown. We here combined short and long-read (nanopore) next-generation sequencing and present the first high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material. We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that map closely together, and complex integration loci that exhibit local amplification of genomic sequences flanking the viral DNA. Sequence analysis suggests that linear patterns are produced during viral replication by integration of defective/linear genomes into host DNA double strand breaks via non-homologous end joining, NHEJ. In contrast, our data strongly suggest that complex integration patterns are mediated by microhomology-mediated break-induced replication, MMBIR. Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, rather than transcription from juxtaposed host promoters. Taken together, our data explain the characteristics of MCPyV integration and may also provide a model for integration of other oncogenic DNA viruses such as papillomaviruses. Author summaryIntegration of viral DNA into the host genome is a key event in the pathogenesis of many virus-induced cancers. One such cancer is Merkel cell carcinoma (MCC), a highly malignant tumor that harbors monoclonally integrated and replication-defective Merkel cell polyomavirus (MCPyV) genomes. Although MCPyV integration sites have been analyzed before, there is very little knowledge of the mechanisms that lead to mutagenesis and integration of viral genomes. We used multiple sequencing technologies and interrogation of chromatin states to perform a comprehensive characterization of MCPyV integration loci. This analysis allowed us to deduce the events that likely precede viral integration. We provide evidence that the mutations which result in the replication defective phenotype are acquired prior to integration and propose that the cellular DNA repair pathways non-homologous end joining (NHEJ) and microhomology-mediated break-induced replication (MMBIR) produce two principal MCPyV integration patterns (simple and complex, respectively). We show that, although MCPyV integrates predominantly in open chromatin regions, viral oncogene expression is independent of host promoters and driven by the viral promotor region. Our findings are important since they can explain the mechanisms of MCPyV integration. Furthermore, our model may also apply to papillomaviruses, another clinically important family of oncogenic DNA viruses.

molecular biology↗

bioRxiv2019

Oncogenic herpesvirus engages the endothelial transcription factors SOX18 and PROX1 to increase viral genome copies and virus production

Kaposi sarcoma (KS) is a tumour of endothelial origin caused by KS herpesvirus (KSHV) infection and suggested to originate from lymphatic endothelial cells (LECs). While KSHV establishes latency in virtually all susceptible cell types, LECs support a spontaneous lytic gene expression program with high viral genome copies and release of infectious virus. Here, we investigated the role of PROX1, SOX18 and COUPTF2, drivers of lymphatic endothelial fate during embryogenesis, in this unique KSHV infection program. We found that these factors were co-expressed in KS tumours with the viral lytic marker K8.1, and that SOX18 and PROX1 regulate KSHV infection via two independent mechanisms. SOX18 binds to the viral origins of replication and its depletion or chemical inhibition significantly reduced the KSHV genome copies in LECs. PROX1 interacts with ORF50, the initiator of the lytic cascade, increases lytic gene expression and virus production and its depletion reduces KSHV spontaneous lytic reactivation. Upon lytic replication, PROX1 binds to the KSHV genome in the promoter region of ORF50 and enhances its transactivation activity. These results demonstrate the importance of two endothelial transcription factors in the regulation of the KSHV life cycle and introduce SOX18 inhibition as a potential, novel therapeutic modality for KS.

microbiology↗

bioRxiv2019

The first days in the life of naïve human B-lymphocytes infected with Epstein-Barr virus

Epstein-Barr virus (EBV) infects and activates resting human B-lymphocytes, reprograms them, induces their proliferation, and establishes a latent infection in them. In established EBV-infected cell lines many viral latent genes are expressed. Their roles in supporting the continuous proliferation of EBV-infected B cells in vitro are known, but their functions in the early, pre-latent phase of infection have not been investigated systematically. In studies during the first eight days of infection using derivatives of EBV with mutations in single genes of EBVs we found only EBNA2 to be essential for activating naive human B-lymphocytes, inducing their growth in cell volume, driving them into rapid cell divisions, and preventing cell death in a subset of infected cells. EBNA-LP, LMP2A and the viral microRNAs have supportive, auxiliary functions, but mutants of LMP1, EBNA3A, EBNA3C, and the noncoding EBER RNAs had no discernable phenotype compared with wild-type EBV. B cells infected with a double mutant of EBNA3A and 3C had an unexpected proliferative advantage and did not regulate the DNA damage response (DDR) of the infected host cell in the pre-latent phase. Even EBNA1 which has very critical long-term functions in maintaining and replicating the viral genomic DNA in established cell lines, was dispensable for the early activation of infected cells. Our findings document that the virus dose is a critical parameter and indicate that EBNA2 governs the infected cells initially and implements a strictly controlled temporal program independent of other viral latent genes. It thus appears that EBNA2 is sufficient to control all requirements for clonal cellular expansion and to reprogram human B-lymphocytes from energetically quiescent to activated cells.\n\nAuthor summaryThe preferred target of Epstein-Barr virus (EBV) are human resting B-lymphocytes. We found that their infection induces a well-coordinated, time-driven program that starts with a substantial increase in cell volume followed by cellular DNA synthesis after three days and subsequent rapid rounds of cell divisions on the next day accompanied by some DNA replication stress (DRS). Two to three days later the cells decelerate and turn into stably proliferating lymphoblast cell lines. With the aid of 16 different recombinant EBV strains we investigated the individual contributions of EBVs multiple latent genes during early B-cell infection and found that many do not exert a detectable phenotype or contribute little to EBVs pre-latent phase. The exception is EBNA2 that is essential in governing all aspects of B-cell reprogramming. EBV relies on EBNA2 to turn the infected B-lymphocytes into proliferating lymphoblasts preparing the infected host cell for the ensuing stable, latent phase of viral infection. In the early steps of B-cell reprogramming viral latent genes other than EBNA2 are dispensable but some, EBNA-LP for example, support the viral program and presumably stabilize the infected cells once viral latency is established.

microbiology↗

bioRxiv2019

Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

Herpesvirus genome replication, capsid assembly and packaging take place in the host cell nucleus. Matured capsids leave the nucleus through a unique envelopment-de-envelopment process at the nuclear membranes called nuclear egress. How assembled and DNA-containing herpesvirus capsids reach the sites of nuclear egress is however still controversially discussed, as host chromatin that marginalizes during infection might constitute a major barrier. For alphaherpesviruses, previous work has suggested that nuclear capsids use active transport mediated by nuclear filamentous actin (F-actin). However, direct evidence for nuclear capsid motility on nuclear F-actin was missing. Our subsequent work did not detect nuclear F-actin associated with motile capsids, but instead found evidence for chromatin remodeling to facilitate passive capsid diffusion. A recent report described that human cyto-megalovirus, a betaherpesvirus, induces nuclear F-actin and that the motor protein myosin V localizes to these structures. Direct evidence of capsid recruitment to these structures and motility on them was however missing. In this study, we tested the functional role of HCMV-induced, nuclear actin assemblies for capsid transport. We did not observe transport events along nuclear F-actin. Instead, reproduction of nuclear F-actin was only possible using strong overexpression of the fluorescent marker LifeAct-mCherry-NLS. Also, two alternative fluo-rescent F-actin markers did not detect F-actin in HCMV-infected cells. Furthermore, single particle tracking of nuclear HCMV capsids showed no indication for active transport, which is in line with previous work on alphaherpesviruses.

microbiology↗

bioRxiv2019

A comparative epigenome analysis of gammaherpesviruses suggests cis-acting sequence features as critical mediators of rapid polycomb recruitment

Latent Kaposi sarcoma-associated herpesvirus (KSHV) genomes rapidly acquire distinct patterns of the activating histone modification H3K4-me3 as well as repressive H3K27-me3 marks, a modification linked to transcriptional silencing by polycomb repressive complexes (PRC). Interestingly, PRCs have recently been reported to restrict viral gene expression in a number of other viral systems, suggesting they may play a broader role in controlling viral chromatin. If so, it is an intriguing possibility that latency establishment may result from viral subversion of polycomb-mediated host responses to exogenous DNA.\n\nTo investigate such scenarios we sought to establish whether rapid repression by PRC constitutes a general hallmark of herpesvirus latency. For this purpose, we performed a comparative epigenome analysis of KSHV and the related murine gammaherpesvirus 68 (MHV-68). We demonstrate that, while latently replicating MHV-68 genomes readily acquire distinct patterns of activation-associated histone modifications upon de novo infection, they fundamentally differ in their ability to efficiently attract H3K27-me3 marks. Statistical analyses of ChIP-seq data from in vitro infected cells as well as in vivo latency reservoirs furthermore suggest that, whereas KSHV rapidly attracts PRCs in a genome-wide manner, H3K27-me3 acquisition by MHV-68 genomes may require spreading from initial seed sites to which PRC are recruited as the result of an inefficient or stochastic recruitment, and that immune pressure may be needed to select for latency pools harboring PRC-silenced episomes in vivo.\n\nUsing co-infection experiments and recombinant viruses, we also show that KSHVS ability to rapidly and efficiently acquire H3K27-me3 marks does not depend on the host cell environment or unique properties of the KSHV-encoded LANA protein, but rather requires specific cis-acting sequence features. We show that the non-canonical PRC1.1 component KDM2B, a factor which binds to unmethylated CpG motifs, is efficiently recruited to KSHV genomes, indicating that CpG island characteristics may constitute these features. In accord with the fact that, compared to MHV-68, KSHV genomes exhibit a fundamentally higher density of CpG motifs, we furthermore demonstrate efficient acquisition of H2AK119-ub by KSHV and H3K36-me2 by MHV-68 (but not vice versa), furthermore supporting the notion that KSHV genomes rapidly attract PRC1.1 complexes in a genome-wide fashion. Collectively, our results suggest that rapid PRC silencing is not a universal feature of viral latency, but that some viruses may rather have adopted distinct genomic features to specifically exploit default host pathways that repress epigenetically naive, CpG-rich DNA.\n\nAuthor SummaryDuring herpesvirus latency, viral genomes persists as partially repressed nuclear episomes which do not express genes required for progeny production. Latently infected cells not only form a reservoir of lifelong persistence but also represent the driving force in cancers associated with tumorigenic herpesviruses such as KSHV. Hence, it is fundamentally important to understand the mechanisms controlling latency. We have shown previously that latent KSHV episomes rapidly acquire H3K27-me3, a histone mark associated with polycomb repressive complexes (PRC). PRCs play a pivotal role in the control of developmental genes but are also involved in the pathogenesis of several tumors. We here investigated whether PRC-repression represents a general feature of herpesvirus latency. By performing side-by-side analyses of KSHV and the related MHV-68 we show that the latter indeed has a fundamentally lower propensity to acquire H3K27-me3, and that KSHVS ability to rapidly attract this mark is most likely the result of a specific sequence composition that promotes recruitment of non-canonical PRC1 (a complex which is important for the regulation of cellular CpG islands). Our results have widespread implications for nuclear DNA viruses and suggest that some viruses have specifically evolved to exploit common host responses to epigenetically naive DNA.

microbiology↗

bioRxiv2018

The landscape of viral associations in human cancers

Potential viral pathogens were systematically investigated in the whole-genome and transcriptome sequencing of 2,656 donors as part of the Pan-Cancer Analysis of Whole Genomes using a consensus approach integrating three independent pathogen detection pipelines. Viruses were detected in 382 genomic and 68 transcriptome data sets. We extensively searched and characterized numerous features of virus-positive cancers integrating various PCAWG datasets. We show the high prevalence of known tumor associated viruses such as EBV, HBV and several HPV types. Our systematic analysis revealed that HPV presence was significantly exclusive with well-known driver mutations in head/neck cancer. A strong association was observed between HPV infection and the APOBEC mutational signatures, suggesting the role of impaired mechanism of antiviral cellular defense as a driving force in the development of cervical, bladder and head neck carcinoma. Viral integration into the host genome was observed for HBV, HPV16, HPV18 and AAV2 and associated with a local increase in copy number variations. The recurrent viral integrations at the TERT promoter were coupled to high telomerase expression uncovering a further mechanism to activate this tumor driving process. High levels of endogenous retrovirus ERV1 expression is linked to worse survival outcome in kidney cancer.

cancer biology↗