Decoding murine cytomegalovirus.

The genomes of both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) were first sequenced over 20 years ago. Similar to HCMV, the MCMV genome had initially been proposed to harbor ≈170 open reading frames (ORFs). More recently, omics approaches revealed HCMV gene expression to be substantially more complex comprising several hundred viral ORFs. Here, we provide a state-of-the art reannotation of lytic MCMV gene expression based on integrative analysis of a large set of omics data. Our data reveal 365 viral transcription start sites (TiSS) that give rise to 380 and 454 viral transcripts and ORFs, respectively. The latter include >200 small ORFs, some of which represented the most highly expressed viral gene products. By combining TiSS profiling with metabolic RNA labelling and chemical nucleotide conversion sequencing (dSLAM-seq), we provide a detailed picture of the expression kinetics of viral transcription. This not only resulted in the identification of a novel MCMV immediate early transcript encoding the m166.5 ORF, which we termed ie4, but also revealed a group of well-expressed viral transcripts that are induced later than canonical true late genes and contain an initiator element (Inr) but no TATA- or TATT-box in their core promoters. We show that viral upstream ORFs (uORFs) tune gene expression of longer viral ORFs expressed in cis at translational level. Finally, we identify a truncated isoform of the viral NK-cell immune evasin m145 arising from a viral TiSS downstream of the canonical m145 mRNA. Despite being ≈5-fold more abundantly expressed than the canonical m145 protein it was not required for downregulating the NK cell ligand, MULT-I. In summary, our work will pave the way for future mechanistic studies on previously unknown cytomegalovirus gene products in an important virus animal model.

Dissecting Herpes Simplex Virus 1-Induced Host Shutoff at the RNA Level.

Herpes simplex virus 1 (HSV-1) induces a profound host shutoff during lytic infection. The virion host shutoff (vhs) protein plays a key role in this process by efficiently cleaving host and viral mRNAs. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in host transcriptional activity. To dissect relative contributions of both mechanisms and elucidate gene-specific host transcriptional responses throughout the first 8 h of lytic HSV-1 infection, we used transcriptome sequencing of total, newly transcribed (4sU-labeled) and chromatin-associated RNA in wild-type (WT) and Δvhs mutant infection of primary human fibroblasts. Following virus entry, vhs activity rapidly plateaued at an elimination rate of around 30% of cellular mRNAs per hour until 8 h postinfection (p.i.). In parallel, host transcriptional activity dropped to 10 to 20%. While the combined effects of both phenomena dominated infection-induced changes in total RNA, extensive gene-specific transcriptional regulation was observable in chromatin-associated RNA and was surprisingly concordant between WT and Δvhs infections. Both induced strong transcriptional upregulation of a small subset of genes that were poorly expressed prior to infection but already primed by H3K4me3 histone marks at their promoters. Most interestingly, analysis of chromatin-associated RNA revealed vhs-nuclease-activity-dependent transcriptional downregulation of at least 150 cellular genes, in particular of many integrin adhesome and extracellular matrix components. This was accompanied by a vhs-dependent reduction in protein levels by 8 h p.i. for many of these genes. In summary, our study provides a comprehensive picture of the molecular mechanisms that govern cellular RNA metabolism during the first 8 h of lytic HSV-1 infection.IMPORTANCE The HSV-1 virion host shutoff (vhs) protein efficiently cleaves both host and viral mRNAs in a translation-dependent manner. In this study, we model and quantify changes in vhs activity, as well as virus-induced global loss of host transcriptional activity, during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were dominated by these two global effects. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and Δvhs infections, confirmed DUX4 as a key transcriptional regulator in HSV-1 infection, and identified vhs-dependent transcriptional downregulation of the integrin adhesome and extracellular matrix components. The latter explained seemingly gene-specific effects previously attributed to vhs-mediated mRNA degradation and resulted in a concordant loss in protein levels by 8 h p.i. for many of the respective genes.

HSV-1-induced disruption of transcription termination resembles a cellular stress response but selectively increases chromatin accessibility downstream of genes.

Lytic herpes simplex virus 1 (HSV-1) infection triggers disruption of transcription termination (DoTT) of most cellular genes, resulting in extensive intergenic transcription. Similarly, cellular stress responses lead to gene-specific transcription downstream of genes (DoG). In this study, we performed a detailed comparison of DoTT/DoG transcription between HSV-1 infection, salt and heat stress in primary human fibroblasts using 4sU-seq and ATAC-seq. Although DoTT at late times of HSV-1 infection was substantially more prominent than DoG transcription in salt and heat stress, poly(A) read-through due to DoTT/DoG transcription and affected genes were significantly correlated between all three conditions, in particular at earlier times of infection. We speculate that HSV-1 either directly usurps a cellular stress response or disrupts the transcription termination machinery in other ways but with similar consequences. In contrast to previous reports, we found that inhibition of Ca2+ signaling by BAPTA-AM did not specifically inhibit DoG transcription but globally impaired transcription. Most importantly, HSV-1-induced DoTT, but not stress-induced DoG transcription, was accompanied by a strong increase in open chromatin downstream of the affected poly(A) sites. In its extent and kinetics, downstream open chromatin essentially matched the poly(A) read-through transcription. We show that this does not cause but rather requires DoTT as well as high levels of transcription into the genomic regions downstream of genes. This raises intriguing new questions regarding the role of histone repositioning in the wake of RNA Polymerase II passage downstream of impaired poly(A) site recognition.

Deciphering the modulation of gene expression by type I and II interferons combining 4sU-tagging, translational arrest and in silico promoter analysis.

Interferons (IFN) play a pivotal role in innate immunity, orchestrating a cell-intrinsic anti-pathogenic state and stimulating adaptive immune responses. The complex interplay between the primary response to IFNs and its modulation by positive and negative feedback loops is incompletely understood. Here, we implement the combination of high-resolution gene-expression profiling of nascent RNA with translational inhibition of secondary feedback by cycloheximide. Unexpectedly, this approach revealed a prominent role of negative feedback mechanisms during the immediate (≤60 min) IFNα response. In contrast, a more complex picture involving both negative and positive feedback loops was observed on IFNγ treatment. IFNγ-induced repression of genes associated with regulation of gene expression, cellular development, apoptosis and cell growth resulted from cycloheximide-resistant primary IFNγ signalling. In silico promoter analysis revealed significant overrepresentation of SP1/SP3-binding sites and/or GC-rich stretches. Although signal transducer and activator of transcription 1 (STAT1)-binding sites were not overrepresented, repression was lost in absence of STAT1. Interestingly, basal expression of the majority of these IFNγ-repressed genes was dependent on STAT1 in IFN-naïve fibroblasts. Finally, IFNγ-mediated repression was also found to be evident in primary murine macrophages. IFN-repressed genes include negative regulators of innate and stress response, and their decrease may thus aid the establishment of a signalling perceptive milieu.

Regulation of autologous immunity to the mouse 5T4 oncofoetal antigen: implications for immunotherapy.

Effective vaccination against tumour-associated antigens (TAA) such as the 5T4 oncofoetal glycoprotein may be limited by the nature of the T cell repertoire and the influence of immunomodulatory factors in particular T regulatory cells (Treg). Here, we identified mouse 5T4-specific T cell epitopes using a 5T4 knock out (5T4KO) mouse and evaluated corresponding wild-type (WT) responses as a model to refine and improve immunogenicity. We have shown that 5T4KO mice vaccinated by replication defective adenovirus encoding mouse 5T4 (Adm5T4) generate potent 5T4-specific IFN-γ CD8 and CD4 T cell responses which mediate significant protection against 5T4 positive tumour challenge. 5T4KO CD8 but not CD4 primed T cells also produced IL-17. By contrast, Adm5T4-immunized WT mice showed no tumour protection consistent with only low avidity CD8 IFN-γ, no IL-17 T cell responses and no detectable CD4 T cell effectors producing IFN-γ or IL-17. Treatment with anti-folate receptor 4 (FR4) antibody significantly reduced the frequency of Tregs in WT mice and enhanced 5T4-specific IFN-γ but reduced IL-10 T cell responses but did not reveal IL-17-producing effectors. This altered balance of effectors by treatment with FR4 antibody after Adm5T4 vaccination provided modest protection against autologous B16m5T4 melanoma challenge. The efficacy of 5T4 and some other TAA vaccines may be limited by the combination of TAA-specific T regs, the deletion and/or alternative differentiation of CD4 T cells as well as the absence of distinct subsets of CD8 T cells.

Integrative functional genomics decodes herpes simplex virus 1.

The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution.

Optimisation of robust singleplex and multiplex droplet digital PCR assays for high confidence mutation detection in circulating tumour DNA.

Liquid biopsies offer the potential to monitor cancer response and resistance to therapeutics in near real-time. However, the plasma cell free DNA (cfDNA) level can be low and the fraction of circulating tumour DNA (ctDNA) bearing a mutation - lower still. Detection of tumour-derived mutations in ctDNA is thus challenging and requires highly sensitive and specific assays. Droplet digital PCR (ddPCR) is a technique that enables exquisitely sensitive detection and quantification of DNA/RNA markers from very limiting clinical samples, including plasma. The Bio-Rad QX200 ddPCR system provides absolute quantitation of target DNA molecules using fluorescent dual-labelled probes. Critical to accurate sample analysis are validated assays that are highly specific, reproducible, and with known performance characteristics, especially with respect to false positives. We present a systematic approach to the development and optimisation of singleplex and multiplex ddPCR assays for the detection of point mutations with a focus on ensuring extremely low false positives whilst retaining high sensitivity. We also present a refined method to determine cfDNA extraction efficiency allowing for more accurate extrapolation of mutational levels in source samples. We have applied these approaches to successfully analyse many ctDNA samples from multiple clinical studies and generated exploratory data of high quality.

High-Resolution Gene Expression Profiling of RNA Synthesis, Processing, and Decay by Metabolic Labeling of Newly Transcribed RNA Using 4-Thiouridine.

Cellular RNA levels are orchestrated by highly regulated processes involving RNA synthesis (transcription), processing (e.g., splicing, polyadenylation, transport), and degradation. Profiling these changes provides valuable information on the regulation of gene expression. Total cellular RNA is a poor template for revealing short-term changes in gene expression, alterations in RNA decay rates, and the kinetics of RNA processing as well as the differentiation thereof. Here, we describe the metabolic labeling and purification of newly transcribed RNA with 4-thiouridine, by which these limitations are overcome.

Widespread disruption of host transcription termination in HSV-1 infection.

Herpes simplex virus 1 (HSV-1) is an important human pathogen and a paradigm for virus-induced host shut-off. Here we show that global changes in transcription and RNA processing and their impact on translation can be analysed in a single experimental setting by applying 4sU-tagging of newly transcribed RNA and ribosome profiling to lytic HSV-1 infection. Unexpectedly, we find that HSV-1 triggers the disruption of transcription termination of cellular, but not viral, genes. This results in extensive transcription for tens of thousands of nucleotides beyond poly(A) sites and into downstream genes, leading to novel intergenic splicing between exons of neighbouring cellular genes. As a consequence, hundreds of cellular genes seem to be transcriptionally induced but are not translated. In contrast to previous reports, we show that HSV-1 does not inhibit co-transcriptional splicing. Our approach thus substantially advances our understanding of HSV-1 biology and establishes HSV-1 as a model system for studying transcription termination.

Metabolic labeling of newly transcribed RNA for high resolution gene expression profiling of RNA synthesis, processing and decay in cell culture.

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated. We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

CD45-CD14 +CD34 + murine bone marrow low-adherent mesenchymal primitive cells preserve multilineage differentiation potential in long-term in vitro culture.

Bone marrow-derived cells have been postulated as a source of multipotent mesenchymal stem cells (MSC). However, the whole fraction of MSC remains heterogeneous and the expansion of primitive subset of these cells is still not well established. Here, we optimized the protocol for propagating the low-adherent subfraction of MSC which results in long-term expansion of population characterized by CD45(-)CD14(+)CD34(+) phenotype along with expression of common MSC markers. We established that the expanded MSC are capable of differentiating into endothelial cells highly expressing angiogenic markers and exhibiting functional properties of endothelium. Moreover, we found these cells to be multipotent and capable of giving rise into cells from neuronal lineages. Interestingly, the expanded MSC form characteristic cellular spheres in vitro indicating primitive features of these cells. In sum, we isolated the novel multipotent subpopulation of CD45(-)CD14(+) CD34(+) bone marrow-derived cells that could be maintained in long-term culture without losing this potential.

CXCR4 mediated chemotaxis is regulated by 5T4 oncofetal glycoprotein in mouse embryonic cells.

5T4 oncofetal molecules are highly expressed during development and upregulated in cancer while showing only low levels in some adult tissues. Upregulation of 5T4 expression is a marker of loss of pluripotency in the early differentiation of embryonic stem (ES) cells and forms an integrated component of an epithelial-mesenchymal transition, a process important during embryonic development and metastatic spread of epithelial tumors. Investigation of the transcriptional changes in early ES differentiation showed upregulation of CXCL12 and down-regulation of a cell surface protease, CD26, which cleaves this chemokine. CXCL12 binds to the widely expressed CXCR4 and regulates key aspects of development, stem cell motility and tumour metastasis to tissues with high levels of CXCL12. We show that the 5T4 glycoprotein is required for optimal functional cell surface expression of the chemokine receptor CXCR4 and CXCL12 mediated chemotaxis in differentiating murine embryonic stem cells and embryo fibroblasts (MEF). Cell surface expression of 5T4 and CXCR4 molecules is co-localized in differentiating ES cells and MEF. By contrast, differentiating ES and MEF derived from 5T4 knockout (KO) mice show only intracellular CXCR4 expression but infection with adenovirus encoding mouse 5T4 restores CXCL12 chemotaxis and surface co-localization with 5T4 molecules. A series of chimeric constructs with interchanged domains of 5T4 and the glycoprotein CD44 were used to map the 5T4 sequences relevant for CXCR4 membrane expression and function in 5T4KO MEF. These data identified the 5T4 transmembrane domain as sufficient and necessary to enable CXCR4 cell surface expression and chemotaxis. Furthermore, some monoclonal antibodies against m5T4 can inhibit CXCL12 chemotaxis of differentiating ES cells and MEF which is not mediated by simple antigenic modulation. Collectively, these data support a molecular interaction of 5T4 and CXCR4 occurring at the cell surface which directly facilitates the biological response to CXCL12. The regulation of CXCR4 surface expression by 5T4 molecules is a novel means to control responses to the chemokine CXCL12 for example during embryogenesis but can also be selected to advantage the spread of a 5T4 positive tumor from its primary site.

Heme oxygenase-1 and the vascular bed: from molecular mechanisms to therapeutic opportunities.

Heme oxygenase-1, an enzyme degrading heme to carbon monoxide, iron, and biliverdin, has been recognized as playing a crucial role in cellular defense against stressful conditions, not only related to heme release. HO-1 protects endothelial cells from apoptosis, is involved in blood-vessel relaxation regulating vascular tone, attenuates inflammatory response in the vessel wall, and participates in blood-vessel formation by means of angiogenesis and vasculogenesis. The latter functions link HO-1 not only to cardiovascular ischemia but also to many other conditions that, like development, wound healing, or cancer, are dependent on neovascularization. The aim of this comprehensive review is to address the mechanisms of HO-1 regulation and function in cardiovascular physiology and pathology and to demonstrate some possible applications of the vast knowledge generated so far. Recent data provide powerful evidence for the involvement of HO-1 in the therapeutic effect of drugs used in cardiovascular diseases. Novel studies open the possibilities of application of HO-1 for gene and cell therapy. Therefore, research in forthcoming years should help to elucidate both the real role of HO-1 in the effect of drugs and the clinical feasibility of HO-1-based cell and gene therapy, creating the effective therapeutic avenues for this refined antioxidant system.