Replicative intermediates of Tomato leaf curl virus and its satellite DNAs

AbstractSeveral plant geminiviruses have been shown recently to utilize both rolling-circle replication (RCR) and recombination-dependent replication (RDR) strategies. A highly specific binding of the viral replication-associated protein (Rep) to its cognate DNA is essential for initiation of viral DNA replication and for the recognition of DNA components of the bipartite geminiviruses of the Begomovirus genus. We have extended the replication analysis to the monopartite Australian Tomato leaf curl virus (ToLCV), its Rep binding deficient mutants, and the satellite DNAs it supports. Analyses of viral DNA by two-dimensional agarose gel electrophoresis after fractionation by single-stranded (ss) DNA-selective cellulose chromatography revealed that DNA intermediates of ToLCV and its mutant were identical. Both RCR and RDR intermediates were identified. New ToLCV DNA forms were observed and characterized as subgenomic topoisomers, heterogeneous open circular double-stranded (ds) DNA, and degradation products. A 1350-nt DNA β satellite associated with the unrelated Cotton leaf curl Multan virus (CLCuMV) was supported by ToLCV and produced intermediates of both RCR and RDR, suggesting that replication strategies of satellites are determined by the helper virus. Replicative intermediates of the 682 nt ToLCV satellite DNA could not be resolved; however, concatemers of up to octamer were detected, together with a field of hybridizing material suggestive of complementary strand replication on heterogeneous circular ssDNA templates

Episomal replication timing of γ-herpesviruses in latently infected cells

AbstractThis study addresses the timing of gammaherpesviral episomal DNA replication with respect to the cell cycle. For the first time we analyzed a rhadinovirus, the prototype Herpesvirus saimiri (HVS), and compared it to the lymphocryptovirus Epstein–Barr virus (EBV). Newly synthesized DNA of latently infected B- or T-cells was first BrdU-labeled; then we sorted the cells corresponding to cell cycle phases G0/1, G2/M, and S (4 fractions S1–S4) and performed anti-BrdU chromatin immunoprecipitation. Next, DNA of different viral gene loci was quantitatively detected together with cellular control genes of known replication time. The sensitive technique is further enhanced by an internal coprecipitation standard for increased precision. Both gammaherpesviruses replicated very early in S-phase, together with cellular euchromatin. Our work suggests that early S-phase DNA replication is a general characteristic of episomal herpesviral genomes

Molecular profiling of single circulating tumor cells with diagnostic intention

Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non-random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC-positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of >90% for successful molecular analysis of high-quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre-existing cells resistant to ERBB2-targeted therapies suggesting ongoing microevolution at late-stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance

Molecular profiling of single circulating tumor cells with diagnostic intention

Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non-random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC-positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of >90% for successful molecular analysis of high-quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre-existing cells resistant to ERBB2-targeted therapies suggesting ongoing microevolution at late-stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance

The Epigenetics of the Herpesvirus saimiri Genome

Herpesviren haben die Eigenschaft, in bestimmten Wirtszellen nach der Primärinfektion einen stabilen Zustand der Latenz zu errichten und sind so in der Lage über lange Zeiträume im Wirtsorganismus zu persistieren. Um die Latenz zu etablieren, werden die meisten viralen Gene stillgelegt. Auf diese Weise kann das Virus einer Immunantwort weitgehend ausweichen. In dieser Arbeit wurde anhand von Herpesvirus saimiri (HVS), einem 2-Herpesvirus aus dem Totenkopfaffen Saimiri sciureus, untersucht, inwieweit das Stilllegen von viralen Genen durch epigenetische Mechanismen reguliert wird. HVS ist in der Lage, humane T-Lymphozyten in vitro zu stabilem Wachstum zu transformieren und verbleibt latent als nicht-integrierendes, zirkuläres und Histon-assoziiertes Episom im Zellkern dieser T-Lymphozyten. Die Identifizierung euchromatischer und heterochromatischer Regionen wurde mit Hilfe von Chromatinimmunpräzipitation (ChIP) und quantitativer PCR analysiert. Das virale Genom wurde auf DNA-Methylierung, auf sechs verschiedene Histonmodifikationen und auf die Inkorporation von transkriptionsspezifischen Histonvarianten hin erforscht. Zum einen wurden die Promotorbereiche einzelner viraler Gene, die repräsentativ für Gene der Latenz bzw. für Gene der sehr frühen, frühen und späten Phase der lytischen Replikation waren, sowie die nicht-kodierenden Wiederholungseinheiten (H-DNA) von HVS betrachtet. Zum anderen wurde die euchromatische Histonacetylierung in hoher Auflösung über das gesamte HVS-Genom hinweg durch das ChIP-on-Chip-Verfahren bestimmt. Die lytischen Promotoren offenbarten - entsprechend ihrem inaktiven Status in humanen T-Zellen - durchweg einen heterochromatischen Phänotyp. Die stabil exprimierten Onkogene stpC und tip und die nicht-kodierende H-DNA waren dagegen mit euchromatischen Histonmarkierungen, also mit „offenem“ Chromatin, assoziiert. Dem äußerst schwach exprimierten orf73-Locus konnte weder ein eu- noch ein heterochromatischer Status zugeordnet werden. Hier wurde stattdessen die Bindung des Chromatin-organisierenden Faktors CTCF nachgewiesen, der sowohl über Chromatindomänen-isolierende als auch Enhancer-blockierende Eigenschaften verfügt. Die Latenz von HVS in humanen T-Zellen gilt als stabiler und irreversibler Zustand. Durch Inkubation mit dem Histondeacetylaseinhibitor Trichostatin A (TSA) ist es hier dennoch gelungen, auf Ebene der Histonacetylierung die Induktion der frühen lytischen Phase nachzuweisen. Nach vierstündiger Inkubation mit TSA waren spezifisch die Promotoren der sehr frühen Gene und etwa die Hälfte der frühen viralen Gene acetyliert, die späten Gene blieben erwartungsgemäß hypoacetyliert. Folgerichtig konnte auch eine deutlich erhöhte Transkription der sehr frühen Gene belegt werden. Diese Daten lassen erkennen, dass epigenetische Mechanismen auch bei Rhadinoviren entscheidend sowohl an der herpesviralen Latenz als auch am Übergang in die lytische Replikation beteiligt sind.All herpesviruses are able to establish latency in suitable host cells after primary infection and persist in the host organisms a lifetime. In order to establish latency, most of the viral genes are silenced enabling the virus to escape an immune response. This study addresses the regulation of viral gene silencing by epigenetic mechanisms using Herpesvirus saimiri (HVS) as a model system. HVS is a 2-herpesvirus isolated from the squirrel monkey Saimiri sciureus and is able to transform human T cells to stable growth in vitro. The virus persists latently as a non-integrating, circular and histone associated episome in the nuclei of those T cells. The identification of euchromatic and heterochromatic regions was analyzed by chromatin immunoprecipitation (ChIP) and quantitative PCR. Selected loci of the viral genome were investigated in terms of DNA methylation, six different histone modifications and incorporation of histone variants. Those selected loci reflected promoters of viral latently expressed genes and immediate early, early and late genes transcribed during lytic replication as well as the non-coding repeats (H-DNA). Furthermore, the whole viral genome was probed for histone acetylation in high resolution by ChIP-on-Chip with a custom tiling microarray. Corresponding to their inactive status in human T cells the lytic promoters consistently revealed a heterochromatic phenotype. In contrast, the stably expressed oncogenes stpC and tip and the H-DNA were associated with euchromatic histone marks, representing ‘open’ chromatin. The very weakly expressed orf73 locus could not be attributed to either chromatin status. Instead the binding of the chomatin organizing factor CTCF, which involves both chromatin insulator and enhancer blocking activity, could be detected in the orf73/orf74 intergenic region. HVS latency in human T lymphocytes is considered a stable and irreversible state. Nevertheless incubation with the histone deacetylase inhibitor Trichostatin A (TSA) resulted in the induction of the early lytic phase on the level of histone acetylation. After four hours of incubation with TSA the promoters of the immediate early and several of the early viral genes were specifically acetylated; as expected, late genes remained unaffected. Consequently, a clear increase of transcription of the immediate early genes was found. These data show that epigenetic mechanisms are crucially involved in both rhadinoviral latency and transition into lytic replication

Histone Modification Pattern of the T-Cellular Herpesvirus Saimiri Genome in Latency

Herpesvirus saimiri (HVS) subgroup C strains are able to growth transform human T lymphocytes in vitro. The stably persisting and nonintegrating HVS episome represents an optimal prerequisite for the investigation of the epigenetic state of latent herpesvirus genomes in vitro. Quantitative chromatin immunoprecipitation experiments using seven different histone acetylation- or methylation-specific antibodies revealed repressive marks at four lytic gene promoters and a variable pattern at the weakly transcribed LANA/orf73 promoter. The constitutive stpC/tip promoter regulating the viral oncoproteins and, more interestingly, the noncoding repetitive H-DNA elements flanking the coding region, showed a permissive chromatin structure. This study provides an appropriate model for the analysis of epigenetic herpesvirus genome modifications and their dynamics in T cells

Single-cell analysis of CTCs with diagnostic precision: Opportunities and challenges for personalized medicine. Review

The generation of variant cancer cells is the major cause of acquired resistance against systemic therapies and consequently, of our inability to cure advanced cancer patients. Circulating tumor cells are gaining increasing clinical attention because they may enable the monitoring cancer progression and adjustment of treatment. In recent years multiple technologies for enrichment, isolation as well as molecular and functional analysis of circulating tumor cells have been developed. Implementation of these technologies in standardized and automated workflows in clinical diagnostics could provide valuable information for real-time monitoring of cancer and eventually new therapeutic strategies for the benefit of patients

Begomoviral Movement Protein Effects in Human and Plant Cells: Towards New Potential Interaction Partners

Geminiviral single-stranded circular DNA genomes replicate in nuclei so that the progeny DNA has to cross both the nuclear envelope and the plasmodesmata for systemic spread within plant tissues. For intra- and intercellular transport, two proteins are required: a nuclear shuttle protein (NSP) and a movement protein (MP). New characteristics of ectopically produced Abutilon mosaic virus (AbMV) MP (MPAbMV), either authentically expressed or fused to a yellow fluorescent protein or epitope tags, respectively, were determined by localization studies in mammalian cell lines in comparison to plant cells. Wild-type MPAbMV and the distinct MPAbMV: reporter protein fusions appeared as curled threads throughout mammalian cells. Co-staining with cytoskeleton markers for actin, intermediate filaments, or microtubules identified these threads as re-organized microtubules. These were, however, not stabilized by the viral MP, as demonstrated by nocodazole treatment. The MP of a related bipartite New World begomovirus, Cleome leaf crumple virus (ClLCrV), resulted in the same intensified microtubule bundling, whereas that of a nanovirus did not. The C-terminal section of MPAbMV, i.e., the protein’s oligomerization domain, was dispensable for the effect. However, MP expression in plant cells did not affect the microtubules network. Since plant epidermal cells are quiescent whilst mammalian cells are proliferating, the replication-associated protein RepAbMV protein was then co-expressed with MPAbMV to induce cell progression into S-phase, thereby inducing distinct microtubule bundling without MP recruitment to the newly formed threads. Co-immunoprecipitation of MPAbMV in the presence of RepAbMV, followed by mass spectrometry identified potential novel MPAbMV-host interaction partners: the peptidyl-prolyl cis-trans isomerase NIMA-interacting 4 (Pin4) and stomatal cytokinesis defective 2 (SCD2) proteins. Possible roles of these putative interaction partners in the begomoviral life cycle and cytoskeletal association modes are discussed

Microfluidic enrichment, isolation and characterization of disseminated melanoma cells from lymph node samples

For the first time in melanoma, novel therapies have recently shown efficacy in the adjuvant therapy setting, which makes companion diagnostics to guide treatment decisions a desideratum. Early spread of disseminated cancer cells (DCC) to sentinel lymph nodes (SLN) is indicative of poor prognosis in melanoma and early DCCs could therefore provide important information about the malignant seed. Here, we present a strategy for enrichment of DCCs from SLN suspensions using a microfluidic device (Parsortix (TM), Angle plc). This approach enables the detection and isolation of viable DCCs, followed by molecular analysis and identification of genetic changes. By optimizing the workflow, the established protocol allows a high recovery of DCC from melanoma patient-derived lymph node (LN) suspensions with harvest rates above 60%. We then assessed the integrity of the transcriptome and genome of individual, isolated DCCs. In LNs of melanoma patients, we detected the expression of melanoma-associated transcripts including MLANA (encoding for MelanA protein), analyzed the BRAF and NRAS mutational status and confirmed the malignant origin of isolated melanoma DCCs by comparative genomic hybridization. We demonstrate the feasibility of epitope-independent isolation of LN DCCs using Parsortix (TM) for subsequent molecular characterization of isolated single DCCs with ample application fields including the use for companion diagnostics or subsequent cellular studies in personalized medicine