Biology subjects

Hennig, T.

Publications and source records attributed to Hennig, T..

At least 19 recordsLinked to original sources

bioRxiv2025

Murine CMV Infection Unmasks Macrophage-Driven Inflammatory Cardiomyopathy in Pkp2, but not in Ttn Mutant Mice

2.BackgroundGenetic cardiomyopathies display variable penetrance and phenotypic expression, highlighting the influence of environmental modulators. Myocarditis, commonly triggered by cardiotropic viruses, overlaps clinically with genetic cardiomyopathies. Consequently, these infections are implicated as secondary factors that accelerate disease onset and progression, yet their precise impact in specific genetic settings remains unexplored. MethodsTo interrogate this, genetic mouse models heterozygous for a mutant allele of desmosomal plakophilin-2 (Pkp2+/-) or sarcomeric titin (Ttn+/-), genes frequently linked to acute myocarditis, were challenged with murine cytomegalovirus (MCMV) to determine how latent infection influences myocardial inflammation, tissue remodeling, and cardiac performance. Integrated experimental approaches, including echocardiography, histology, flow cytometry, single-cell RNA sequencing, as well as cytokine and kinome analyses, defined immune and signaling responses in infected versus non-infected hearts. ResultsAcute, MCMV-induced viral myocarditis and subsequent latent MCMV infection unmasked early disease onset in Pkp2+/- animals, leading to progressive systolic impairment, whereas in Ttn+/- mice cardiac structure and function remained preserved throughout infection. Cardiac immune profiling uncovered infection- and genotype-specific divergence: both genetic models showed a stable myocardial effector-memory CD8+ T-cell response to MCMV, but only Pkp2+/- hearts recruited additional Ly6C+ CCR2+ monocytes and macrophages with distinct inflammatory signatures. In the absence of infection, Pkp2 insufficiency initiated subclinical CCL2 secretion and subsequent recruitment of CCR2+ cells, reflecting early immune activation preceding age-associated functional and structural decline. At this stage, cytokine and kinase evaluations indicated a balance between proinflammatory and compensatory signals. However, with aging or following MCMV challenge, this balance shifted towards persistent inflammation, evidenced by chronic upregulation of cytokines and activation of signaling pathways, which ultimately led to adverse effects and myocardial dysfunction. ConclusionsManifestation of genetic cardiomyopathies depends on interactions between inherited susceptibility and environmental stressors. Here, we show that cytomegalovirus infection intensifies inflammation in PKP2-related cardiomyopathy. In contrast, TTN-linked cardiomyopathy does not exhibit increased inflammation under the same conditions. For individuals carrying desmosomal variants, infection control and tailored anti-inflammatory strategies may attenuate or delay disease manifestation and progression.

immunology↗

bioRxiv2024

Identifying Modulators of Cellular Responses by Heterogeneity-sequencing

The response of individual cells to drug treatment, virus infections or other molecular stimuli is highly heterogeneous and depends on the cells initial state. Library preparation for single-cell transcriptomics is destructive, precluding a direct comparison between the initial state and the stimulus outcome. Consequently, current methods are restricted to identifying correlative associations rather than resolving causal drivers of heterogeneous outcomes. We developed Heterogeneity-seq, which combines single-cell RNA-seq with metabolic RNA labeling (scSLAM-seq) and double machine learning to overcome this limitation. By leveraging simultaneous measurements of unlabeled and labeled RNA in individual cells, Heterogeneity-seq uncovers the transition from pre-stimulated cell states to distinct stimulation outcomes across thousands of cells. These links enable the identification of factors that causally govern heterogeneous cellular responses. We used Heterogeneity-seq to identify both known and novel genes that drive responses to drug treatment, as well as pro- and antiviral host factors governing cytomegalovirus infection.

bioinformatics↗

bioRxiv2024

HSV-1 infection induces a downstream-shift of +1 nucleosomes

Herpes simplex virus 1 (HSV-1) infection induces a loss of host transcriptional activity and widespread disruption of host transcription termination, which leads to an induction of open chromatin downstream of genes. In this study, we show that lytic HSV-1 infection also leads to an extension of chromatin accessibility at promoters into downstream regions. This is most prominent for highly expressed genes and independent of the immediate-early proteins ICP0, ICP22, and ICP27 and the virion host shutoff protein vhs. ChIPmentation of the noncanonical histone variant H2A.Z, which is strongly enriched at +1 and -1 nucleosomes, indicated that these chromatin accessibility changes are linked to a downstream shift of +1 nucleosomes. In yeast, downstream shifts of +1 nucleosomes are induced by RNA Polymerase II (Pol II) degradation. Accordingly, irreversible depletion of Pol II from genes in human cells using -amanitin altered +1 nucleosome positioning similar to lytic HSV-1 infection. Consequently, treatment with phosphonoacetic acid (PAA) and knockout of ICP4, which both prevent viral DNA replication and alleviate the loss of Pol II from host genes, largely abolished the downstream extension of accessible chromatin in HSV-1 infection. In the absence of viral DNA replication, doxycycline-induced expression of ICP27, which redirects Pol II from gene bodies into intergenic regions by disrupting transcription termination, induced an attenuated effect that was further enhanced by co-expression of ICP22. In summary, our study provides strong evidence that HSV-1-induced depletion of Pol II from the host genome leads to a downstream shift of +1 nucleosomes at host promoters. ImportanceLytic herpes simplex virus 1 (HSV-1) infection leads to a profound host transcription shutoff. Loss of RNA Polymerase II (Pol II) in yeast has previously been shown to relax +1 nucleosome positioning to more thermodynamically favorable sites downstream of transcription start sites. Here, we show that a similar phenomenon is likely at play in lytic HSV-1 infection. Sequencing of accessible chromatin revealed a widening of nucleosome-free regions at host promoters into downstream regions. By mapping genome-wide positions of the noncanonical histone variant H2A.Z enriched at +1 and -1 nucleosomes, we demonstrate a downstream shift of +1 nucleosomes for most cellular genes in lytic HSV-1 infection. As chemical depletion of Pol II from genes also leads to a downstream shift of +1 nucleosomes in human cells, changes in chromatin architecture at promoters in HSV-1 infection are likely a consequence of HSV-1-induced loss of Pol II activity from the host genome.

bioinformatics↗

bioRxiv2024

Building Blocks of Understanding: Constructing a Reverse Genetics Platform for studying determinants of SARS-CoV-2 replication.

To better understand viral pathogenesis, host-virus interactions, and potential therapeutic interventions, the development of robust reverse genetics systems for SARS-CoV-2 is crucial. Here, we present a reverse genetics platform that enables the efficient manipulation, assembly, and rescue of recombinant SARS-CoV-2. The versatility of our reverse genetics system was demonstrated by generating recombinant SARS-CoV-2 viruses. We used this system to generate N501Y and Y453F spike protein mutants. Characterization studies revealed distinct phenotypic effects, impact on viral fitness, cell binding, and replication kinetics. We also investigated a recently discovered priming site for NSP9, which is postulated to produce a short RNA antisense leader sequence. By introducing the U76G mutation into the 5UTR, we show that this priming site is necessary for the correct production of genomic and subgenomic RNAs, and also for efficient viral replication. In conclusion, our developed reverse genetics system provides a robust and adaptable platform for the efficient generation of recombinant SARS-CoV-2 viruses for their comprehensive characterization. Significance statementIn this study, we present a versatile reverse genetics platform facilitating the efficient manipulation, assembly, and rescue of recombinant SARS-CoV-2. Demonstrating its adaptability, we successfully engineered N501Y and Y453F spike protein mutants, each exhibiting distinct phenotypic effects on viral fitness, cell binding, and replication kinetics. We also investigated a novel negative sense priming site for NSP9, demonstrating a role in RNA production and viral replication. This straightforward reverse genetic system is therefore a powerful tool to generate recombinant viruses for advancing our understanding of SARS-CoV-2 biology.

microbiology↗

bioRxiv2023

Correcting 4sU induced quantification bias in nucleotide conversion RNA-seq data

Nucleoside analogues like 4-thiouridine (4sU) are used to metabolically label newly synthesized RNA. Chemical conversion of 4sU before sequencing induces T-to-C mismatches in reads sequenced from labelled RNA, allowing to obtain total and labelled RNA expression profiles from a single sequencing library. Cytotoxicity due to extended periods of labelling or high 4sU concentrations has been described, but the effects of extensive 4sU labelling on expression estimates from nucleotide conversion RNA-seq have not been studied. Here, we performed nucleotide conversion RNA-seq with escalating doses of 4sU with short-term labelling (1h) and over a progressive time course (up to 2h) in different cell lines. With high concentrations or at later time points, expression estimates were biased in an RNA half-life dependent manner. We show that bias arose by a combination of reduced mappability of reads carrying multiple conversions, and a global, unspecific underrepresentation of labelled RNA due to impaired reverse transcription efficiency and potentially global reduction of RNA synthesis. We developed a computational tool to rescue unmappable reads, which performed favourably compared to previous read mappers, and a statistical method, which could fully remove remaining bias. All methods developed here are freely available as part of our GRAND-SLAM pipeline and grandR package.

bioinformatics↗

bioRxiv2023

The Cytomegalovirus M35 Protein Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes by Direct Promoter Binding

Induction of type I interferon (IFN) gene expression is among the first lines of cellular defence a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system. M35 localizes to the nucleus and interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35s function. Using electrophoretic mobility shift assays (EMSA), we demonstrate that purified M35 protein specifically binds to the regulatory DNA element that governs transcription of the first type I IFN gene induced in non-immune cells, Ifnb1. Determination of M35s crystal structure combined with reverse genetics revealed that homodimerisation is a key feature for M35s immunomodulatory activity. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signalling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signalling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labelled transcripts (SLAM-seq), and assessed M35s global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically down-regulated basal expression of IRF3-dependent genes, and during MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1. Our results suggest that M35-DNA binding directly antagonises gene induction by IRF3 and impairs the antiviral response more broadly than formerly recognised. ImportanceReplication of the ubiquitous human cytomegalovirus (CMV) in healthy individuals mostly goes unnoticed, but can impair foetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes. Unrelated proteins from other herpesviruses employ the same mechanism, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.

molecular biology↗

bioRxiv2022

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 {approx}170 open reading frames (ORFs). More recently, omics approaches revealed HCMV gene expression to be substantially more complex comprising several hundreds of translated 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 363 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 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 {approx}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. Author summaryWe conducted a comprehensive characterization and reannotation of murine cytomegalovirus (MCMV) gene expression during lytic infection in murine fibroblasts using an integrative multi-omics approach. This unveiled hundreds of novel transcripts that explained the expression of close to 300 so far unknown viral open reading frames (ORFs). Interestingly, small viral ORFs (sORFs) were amongst the most highly expressed viral gene products and thus presumably encode for important viral microproteins of unknown function. However, we also show that sORFs located upstream of larger ORFs tune the expression of the downstream ORFs at the level of translation. We classified viral transcription start sites (TiSS) based on their expression kinetics obtained by a new combination of metabolic RNA labelling with transcription start sites profiling. This not only identified a so far unknown viral immediate-early transcript (ie4, m166.5 RNA) but also revealed a novel class of viral late transcripts that are expressed later than canonical true late genes and lack TATA box-like motifs. We exemplify for the m145 locus how so far unknown TiSS give rise to abundantly expressed truncated viral proteins. In summary, we provide a state-of-the-art annotation of an important model virus, which will be instrumental for future studies on CMV biology, immunology and pathogenesis.

microbiology↗

bioRxiv2022

HSV-1 infection induces a downstream shift of promoter-proximal pausing linked to H2A.Z-containing +1 nucleosomes

Herpes simplex virus 1 (HSV-1) infection exerts a profound shut-off of host gene expression at multiple levels. Recently, HSV-1 infection was reported to also impact promoter-proximal RNA Polymerase II (Pol II) pausing, a key step in the eukaryotic transcription cycle, with decreased and increased Pol II pausing observed for activated and repressed genes, respectively. Here, we demonstrate that HSV-1 infection induces more complex alterations in promoter-proximal pausing than previously suspected for the vast majority of cellular genes. While pausing is generally retained, it is shifted to more downstream and less well-positioned sites for most host genes. We exclude that this is an artefact of alternative de novo transcription initiation at downstream sites or read-in transcription originating from disruption of transcription termination for upstream genes. Use of downstream 2nd pause sites associated with +1 nucleosomes was previously observed upon NELF depletion. However, downstream shifts of Pol II pausing upon HSV-1 infection are much more pronounced than observed upon NELF depletion. Thus, our study reveals a novel aspect in which HSV-1 infection fundamentally reshapes host transcriptional processes with implications for our understanding of maintenance of promoter-proximal Pol II pausing in eukaryotic cells.

molecular biology↗

bioRxiv2022

Is evolutionary conservation a useful predictor for cancer long noncoding RNAs? Insights from the Cancer lncRNA Census 3

Evolutionary conservation is a measure of gene functionality that is widely used to prioritise long noncoding RNAs (lncRNA) in cancer research. Intriguingly, while updating our Cancer LncRNA Census, we observed an inverse relationship between year of discovery and evolutionary conservation. This observation is specific to cancer over other diseases, implying a sampling bias in selection of lncRNA candidates and casting doubt on the value of evolutionary metrics for prioritisation of cancer-related lncRNAs.

cancer biology↗

bioRxiv2022

Data-Driven Design of Protein-Derived Peptide Multiplexes for Biomimetic Detection of Exhaled Breath VOC Profiles

Exhaled human breath contains a rich mixture of volatile organic compounds (VOCs) whose concentration can vary in response to disease or other stressors. Using simulated odorant-binding proteins (OBPs) and machine learning methods, we designed a multiplex of short VOC- and carbon-binding peptide probes that detect the characteristic "VOC fingerprint". Specifically, we target VOCs associated with COVID-19 in a compact, molecular sensor array that directly transduces vapor composition into multi-channel electrical signals. Rapidly synthesizable, chimeric VOC- and solid-binding peptides were derived from selected OBPs using multi-sequence alignment with protein database structures. Selective peptide binding to targeted VOCs and sensor surfaces was validated using surface plasmon resonance spectroscopy and quartz crystal microbalance. VOC sensing was demonstrated by peptide-sensitized, exposed-channel carbon nanotube transistors. The data-to-device pipeline enables the development of novel devices for non-invasive monitoring, diagnostics of diseases, and environmental exposures assessment.

bioengineering↗

bioRxiv2021

HSV-1 and influenza infection induce linear and circular splicing of the long NEAT1 isoform

The herpes simplex virus 1 (HSV-1) virion host shut-off (vhs) protein cleaves both cellular and viral mRNAs by a translation-initiation-dependent mechanism. Here, we show that vhs-mediated degradation of mRNAs leads to an accumulation of circular RNAs (circRNAs) relative to linear mRNAs during HSV-1 infection. Strikingly, we found that circular splicing of the long isoform (NEAT1_2) of the nuclear paraspeckle assembly transcript 1 (NEAT1) was massively induced during HSV-1 infection. In contrast to other circRNAs, induction of the NEAT1_2 circRNA was independent of vhs and occurred while NEAT1_2 was still bound to the chromatin. This was associated with induction of linear splicing of NEAT1_2 both within and downstream of the circRNA. NEAT1_2 splicing was absent in uninfected cells but can be induced by ectopic co-expression of the HSV-1 immediate-early proteins ICP22 and ICP27. Interestingly, NEAT1_2 circular and linear splicing was also up-regulated in influenza A virus (IAV) infection but absent in stress conditions, which disrupt transcription termination similar to but not by the same mechanisms as HSV-1 and IAV infection. Large-scale analysis of published RNA-seq data uncovered induction of NEAT1_2 splicing in cancer cells upon inhibition or knockdown of cyclin-dependent kinase 7 (CDK7) or the MED1 subunit of the Mediator complex phosphorylated by CDK7. Finally, CDK7 inhibition also disrupted transcription termination, highlighting a possible link between disruption of transcription termination and NEAT1_2 splicing.

bioinformatics↗

bioRxiv2021

The Epstein-Barr virus ubiquitin deconjugase BPLF1 regulates the activity of Topoisomerase II during virus replication

Topoisomerases are essential for the replication of herpesviruses but the mechanisms by which the viruses hijack the cellular enzymes are largely unknown. We found that topoisomerase-II (TOP2) is a substrate of the Epstein-Barr virus (EBV) ubiquitin deconjugase BPLF1. BPLF1 selectively inhibited the ubiquitination of TOP2 following treatment with topoisomerase poisons, interacted with TOP2 and TOP2{beta} in co-immunoprecipitation and in vitro pull-down, stabilized Etoposide-trapped TOP2 cleavage complexes (TOP2cc) and promoted TOP2 SUMOylation, which halted the DNA-damage response and reduced Etoposide toxicity. Induction of the productive virus cycle promoted the accumulation of TOP2{beta}cc, enhanced TOP2{beta} SUMOylation, and reduced Etoposide toxicity in lymphoblastoid cell lines carrying recombinant EBV encoding the active enzyme. Attenuation of this phenotype upon expression of a catalytic mutant BPLF1-C61A impaired viral DNA synthesis and virus release. These findings highlight a previously unrecognized function of BPLF1 in promoting non-proteolytic pathways for TOP2cc debulking that favor cell survival and virus production.

microbiology↗

bioRxiv2021

pUL36 de-ubiquitinase activity augments both the initiation and progression of lytic virus infection in IFN-primed cells

The conserved, structural HSV-1 tegument protein pUL36 is essential for both virus entry and assembly. While its N-terminal de-ubiquitinase (DUB) activity is dispensable for infection in cell culture, it is required for efficient virus spread in vivo by acting as a potent viral immune evasin. Here, we show that the pUL36 DUB activity was required to overcome interferon-(IFN)-mediated suppression of both plaque initiation and progression to productive infection. Immediately upon virus entry, incoming tegument-derived pUL36-DUB activity helped the virus to escape intrinsic antiviral resistance and efficiently initiate lytic virus replication in IFN-primed cells. Subsequently, de novo expressed pUL36-DUB augmented the efficiency of productive infection and virus yield. Interestingly, removal of IFN shortly after inoculation only resulted in a partial rescue of plaque formation, indicating that an IFN-induced defense mechanism eliminates invading virus particles unless counteracted by pUL36-DUB activity. Taken together, we demonstrated that the pUL36 DUB disarms IFN-induced antiviral responses at two levels, namely, to protect the infectivity of invading virus as well as to augment productive virus replication in IFN-primed cells. Author SummaryHSV-1 is an ubiquitous human pathogen that is responsible for common cold sores but may also cause life-threatening disease. pUL36 is an essential and conserved protein of infectious herpesvirus virions with a unique de-ubiquitinating (DUB) activity. The pUL36 DUB is dispensable for efficient virus infection in cell culture but represents an important viral immune evasin in vivo. Here, we showed that tegument-derived DUB activity delivered by the invading virus particles is required to overcome IFN-induced host resistance and to initiate efficient lytic infection. De novo expressed pUL36 DUB subsequently augments productive infection and virus yield. These data indicate that the pUL36 DUB antagonizes the activity of yet unidentified IFN-inducible E3 ligases to facilitate productive infection at multiple levels. Our findings underscore the therapeutic potential of targeting conserved herpesvirus DUBs to prevent or treat herpesvirus disease.

microbiology↗

bioRxiv2020

Concatemeric Broccoli reduces mRNA stability, aggregates and induces p-body formation

Fluorogenic aptamers are an alternative to established methodology for real-time imaging of RNA transport and dynamics. We developed Broccoli-aptamer concatemers ranging from 4 to 128 substrate-binding site repeats and characterized their behavior fused to an mCherry-coding mRNA in transient transfection, stable expression, and in recombinant cytomegalovirus infection. Concatemerization of substrate-binding sites increased Broccoli fluorescence up to a concatemer length of 16 copies, upon which fluorescence did not increase and mCherry signals declined. This was due to the combined effects of RNA aptamer aggregation, a nuclear export defect and reduced RNA stability. Unfortunately, both cellular and cytomegalovirus genomes were unable to maintain and express high Broccoli concatemer copy numbers, possibly due to recombination events. Overexpression of Broccoli-tagged mRNA led to the formation of p-bodies. However, Broccoli RNAs did not localize to these sites. Interestingly, negative effects of Broccoli concatemers could be partially rescued by introducing linker sequences in between Broccoli repeats warranting further studies. Finally, we show that even though substrate-bound Broccoli is easily photobleached, it can still be utilized in live-cell imaging by adapting a time-lapse imaging protocol.

microbiology↗

bioRxiv2020

Mechanism and consequences of herpes simplex virus 1-mediated regulation of host mRNA alternative polyadenylation

Eukaryotic gene expression is extensively regulated by cellular stress and pathogen infections. We have previously shown that herpes simplex virus 1 (HSV-1) and several cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes and that the viral immediate early factor ICP27 plays an important role in HSV-1-induced DoTT. Here, we show that HSV-1 infection also leads to widespread changes in alternative polyadenylation (APA) of host mRNAs. In the majority of cases, polyadenylation shifts to upstream poly(A) sites (PAS), including many intronic PAS. Mechanistically, ICP27 contributes to HSV-1-mediated APA regulation. HSV-1- and ICP27-induced activation of intronic PAS is sequence-dependent and does not involve general inhibition of U1 snRNP. HSV1-induced intronic polyadenylation is accompanied by early termination of RNAPII. Finally, HSV-1-induced mRNAs polyadenylated at intronic PAS are exported into the cytoplasm while APA isoforms with extended 3 UTRs are sequestered in the nuclei, both preventing the expression of the full-length gene products. Together with other recent studies, our results suggest that viral infection and cellular stresses induce a multi-faceted host shutoff response that includes DoTT and changes in APA profiles.

molecular biology↗

bioRxiv2020

The zinc finger antiviral protein ZAP destabilises viral transcripts and restricts cytomegalovirus

Interferon-stimulated gene products (ISGs) play a crucial role in early infection control. The ISG zinc finger CCCH-type antiviral protein 1 (ZAP/ZC3HAV1) antagonises several RNA viruses by binding to CG-rich RNA sequences, whereas its effect on DNA viruses is largely unknown. Here, we decipher the role of ZAP in the context of human cytomegalovirus (HCMV) infection, a {beta}-herpesvirus that is associated with high morbidity in immunosuppressed individuals and newborns. We show that expression of the two major isoforms of ZAP, the long (ZAP-L) and short (ZAP-S), is induced during HCMV infection and that both negatively affect HCMV replication. Transcriptome and proteome analyses demonstrated that the expression of ZAP decelerates the progression of HCMV infection. SLAM-sequencing revealed that ZAP restricts HCMV at early stages of infection by destabilising a distinct subset of viral transcripts with low CG content. In summary, this report provides evidence of an important antiviral role for ZAP in host defense against HCMV infection and highlights its differentiated function during DNA virus infection.

molecular biology↗

bioRxiv2020

Dissecting HSV-1-induced host shut-off at RNA level

Herpes simplex virus 1 (HSV-1) installs a profound host shut-off during lytic infection. The virion host shut-off (vhs) protein plays a key role in this process by efficiently cleaving both host and viral mRNAs in a translation-initiation-dependent manner. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in transcriptional activity of the host genome. Both mechanisms have tremendous impact on the RNA expression profile of the infected cells. To dissect their relative contributions and elucidate gene-specific host transcriptional responses throughout the first 8h of lytic HSV-1 infection, we here employed RNA-seq of total, newly transcribed (4sU-labelled) and chromatin-associated RNA in wild-type (WT) and {Delta}vhs 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 8h p.i. In parallel, host transcriptional activity dropped down to 10-20%. 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. This was surprisingly concordant between WT HSV-1 and its {Delta}vhs mutant and at least in parts mediated by the embryonic transcription factor DUX4. Furthermore, both WT and {Delta}vhs infection induced strong transcriptional up-regulation of a small subset of genes. Most of these were either poorly or not at all 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 down-regulation of at least 150 cellular genes, in particular of many genes encoding integrin adhesome and extracellular matrix components. This was accompanied by a vhs-dependent reduction in protein levels by 8h 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 8h of lytic HSV-1 infection. Author SummaryThe HSV-1 virion host shut-off (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 the virus-induced global loss of host transcriptional activity during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were found to be predominantly shaped by these two global processes rather than gene-specific regulation. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and{Delta} vhs infection, confirmed DUX4 as a key transcriptional regulator in HSV-1 infection and depicted vhs-dependent, transcriptional down-regulation of the integrin adhesome and extracellular matrix. The latter explained some of the gene-specific effects previously attributed to vhs-mediated mRNA degradation and resulted in a concordant loss in protein levels by 8h p.i. for many of the respective genes.

microbiology↗

bioRxiv2019

Integrative functional genomics decodes herpes simplex virus 1

Since the genome of herpes simplex virus 1 (HSV-1) was first sequenced more than 30 years ago, its predicted 80 genes have been intensively studied. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identified a total of 201 viral transcripts and 284 open reading frames (ORFs) including all known and 46 novel large ORFs. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of novel viral ORFs as well as N-terminal extensions (NTEs) and truncations thereof. We show that key viral regulators and structural proteins possess NTEs, which initiate from non-canonical start codons and govern subcellular protein localization and packaging. We validated a novel non-canonical large spliced ORF in the ICP0 locus and identified a 93 aa ORF overlapping ICP34.5 that is thus also deleted in the FDA-approved oncolytic virus Imlygic. Finally, we extend the current nomenclature to include all novel viral gene products. Taken together, this work provides a valuable resource for future functional studies, vaccine design and oncolytic therapies.

microbiology↗