Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent of several malignancies of endothelial and B-cell origin. The fact that latently infected tumor cells in these malignancies do not ...express classical viral oncogenes suggests that pathogenesis of KSHV-associated disease results from multistep processes that, in addition to constitutive viral gene expression, may require accumulation of cellular alterations. Heritable changes of the epigenome have emerged as an important co-factor that contributes to the pathogenesis of many non-viral cancers. Since KSHV encodes a number of factors that directly or indirectly manipulate host cell chromatin, it is an intriguing possibility that epigenetic reprogramming also contributes to the pathogenesis of KSHV-associated tumors. The fact that heritable histone modifications have also been shown to regulate viral gene expression programs in KSHV-infected tumor cells underlines the importance of epigenetic control during latency and tumorigenesis. We here review what is presently known about the role of epigenetic regulation of viral and host chromatin in KSHV infection and discuss how viral manipulation of these processes may contribute to the development of KSHV-associated disease.
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.
We describe a multifactorial investigation of a SARS‐CoV‐2 outbreak in a large meat processing complex in Germany. Infection event timing, spatial, climate and ventilation conditions in the ...processing plant, sharing of living quarters and transport, and viral genome sequences were analyzed. Our results suggest that a single index case transmitted SARS‐CoV‐2 to co‐workers over distances of more than 8 m, within a confined work area in which air is constantly recirculated and cooled. Viral genome sequencing shows that all cases share a set of mutations representing a novel sub‐branch in the SARS‐CoV‐2 C20 clade. We identified the same set of mutations in samples collected in the time period between this initial infection cluster and a subsequent outbreak within the same factory, with the largest number of confirmed SARS‐CoV‐2 cases in a German meat processing facility reported so far. Our results indicate climate conditions, fresh air exchange rates, and airflow as factors that can promote efficient spread of SARS‐CoV‐2 via long distances and provide insights into possible requirements for pandemic mitigation strategies in industrial workplace settings.
Synopsis
There has been considerable debate about the factors contributing to SARS‐CoV‐2 outbreaks in food processing facilities around the world. This multifactorial investigation of an outbreak in a German meat processing plant shows that transmission occurred in a confined working area over long distances.
Analysis of infection event timing, spatial, climate and ventilation conditions, living quarters and transport, and viral genome sequences suggests a super spreading event that originated from a single employee.
Infections among workers over a distance of 8 m from the index case suggest aerosol transmission of SARS‐CoV-2.
The facilities’ environmental conditions such as low temperature, low air exchange rates, and constant air recirculation, together with relatively close distance between workers and demanding physical work, may have promoted efficient aerosol transmission.
In contrast to work‐related exposure, shared apartments, bedrooms, or carpools appear not to have played a major role in the initial outbreak.
Viral genome sequencing reveals a characteristic set of mutations that was also observed in samples collected during a later, much larger outbreak occurring in the same processing plant.
There has been considerable debate about the factors contributing to SARS‐CoV‐2 outbreaks in food processing facilities around the world. This multifactorial investigation of an outbreak in a German meat processing plant shows that transmission occurred in a confined working area over long distances.
We have developed an approach to identify microRNAs (miRNAs) that is based on bioinformatics and array-based technologies, without the use of cDNA cloning. The approach, designed for use on genomes ...of small size (<2 Mb), was tested on cells infected by either of two lymphotropic herpesviruses, KSHV and EBV. The viral genomes were scanned computationally for pre-miRNAs using an algorithm (VMir) we have developed. Candidate hairpins suggested by this analysis were then synthesized as oligonucleotides on microarrays, and the arrays were hybridized with small RNAs from infected cells. Candidate miRNAs that scored positive on the arrays were then subjected to confirmatory Northern blot analysis. Using this approach, 10 of the known KSHV pre-miRNAs were identified, as well as a novel pre-miRNA that had earlier escaped detection. This method also led to the identification of seven new EBV-encoded pre-miRNAs; by using additional computational approaches, we identified a total of 18 new EBV pre-miRNAs that produce 22 mature miRNA molecules, thereby more than quadrupling the total number of hitherto known EBV miRNAs. The advantages and limitations of the approach are discussed.
Kaposi sarcoma-associated (KS-associated) herpesvirus (KSHV) infection is linked to the development of both KS and several lymphoproliferative diseases. In all cases, the resulting tumor cells ...predominantly display latent viral infection. KS tumorigenesis requires ongoing lytic viral replication as well, however, for reasons that are unclear but have been suggested to involve the production of angiogenic or mitogenic factors by lytically infected cells. Here we demonstrate that proliferating cells infected with KSHV in vitro display a marked propensity to segregate latent viral genomes, with only a variable but small subpopulation being capable of stable episome maintenance. Stable maintenance is not due to the enhanced production of viral or host trans-acting factors, but is associated with cis-acting, epigenetic changes in the viral chromosome. These results indicate that acquisition of stable KSHV latency is a multistep process that proceeds with varying degrees of efficiency in different cell types. They also suggest an additional role for lytic replication in sustaining KS tumorigenesis: namely, the recruitment of new cells to latency to replace those that have segregated the viral episome.
Cellular and viral microRNAs (miRNAs) are involved in many different processes of key importance and more than 10,000 miRNAs have been identified so far. In general, relatively little is known about ...their biological functions in mammalian cells because their phenotypic effects are often mild and many of their targets still await identification. The recent discovery that Epstein-Barr virus (EBV) and other herpesviruses produce their own, barely conserved sets of miRNAs suggests that these viruses usurp the host RNA silencing machinery to their advantage in contrast to the antiviral roles of RNA silencing in plants and insects. We have systematically introduced mutations in EBV's precursor miRNA transcripts to prevent their subsequent processing into mature viral miRNAs. Phenotypic analyses of these mutant derivatives of EBV revealed that the viral miRNAs of the BHRF1 locus inhibit apoptosis and favor cell cycle progression and proliferation during the early phase of infected human primary B cells. Our findings also indicate that EBV's miRNAs are not needed to control the exit from latency. The phenotypes of viral miRNAs uncovered by this genetic analysis indicate that they contribute to EBV-associated cellular transformation rather than regulate viral genes of EBV's lytic phase.
The biological relevance of extracellular vesicles (EV) in intercellular communication has been well established. Thus far, proteins and RNA were described as main cargo. Here, we show that EV ...released from human bone marrow derived mesenchymal stromal cells (BM-hMSC) also carry high-molecular DNA in addition. Extensive EV characterization revealed this DNA mainly associated with the outer EV membrane and to a smaller degree also inside the EV. Our EV purification protocol secured that DNA is not derived from apoptotic or necrotic cells. To analyze the relevance of EV-associated DNA we lentivirally transduced Arabidopsis thaliana-DNA (A.t.-DNA) as indicator into BM-hMSC and generated EV. Using quantitative polymerase chain reaction (qPCR) techniques we detected high copy numbers of A.t.-DNA in EV. In recipient hMSC incubated with tagged EV for two weeks we identified A.t.-DNA transferred to recipient cells. Investigation of recipient cell DNA using quantitative PCR and verification of PCR-products by sequencing suggested stable integration of A.t.-DNA. In conclusion, for the first time our proof-of-principle experiments point to horizontal DNA transfer into recipient cells via EV. Based on our results we assume that eukaryotic cells are able to exchange genetic information in form of DNA extending the known cargo of EV by genomic DNA. This mechanism might be of relevance in cancer but also during cell evolution and development.
MicroRNAs (miRNAs) are small (∼ 22-nucleotide) RNAs that in lower organisms serve important regulatory roles in development and gene expression, typically by forming imperfect duplexes with target ...messenger RNAs. miRNAs have also been described in mammalian cells and in infections with Epstein-Barr virus (EBV), but the function of most of them is unknown. Although one EBV miRNA probably altered the processing of a viral mRNA, the regulatory significance of this event is uncertain, because other transcripts exist that can supply the targeted function. Here we report the identification of miRNAs encoded by simian virus 40 (SV40) and define their functional significance for viral infection. SVmiRNAs accumulate at late times in infection, are perfectly complementary to early viral mRNAs, and target those mRNAs for cleavage. This reduces the expression of viral T antigens but does not reduce the yield of infectious virus relative to that generated by a mutant lacking SVmiRNAs. However, wild-type SV40-infected cells are less sensitive than the mutant to lysis by cytotoxic T cells, and trigger less cytokine production by such cells. Thus, viral evolution has taken advantage of the miRNA pathway to generate effectors that enhance the probability of successful infection.
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. To 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. Using co-infection experiments and recombinant viruses, we also show that KSHV's 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.
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