Biology subjects

Whisnant, A. W.

Publications and source records attributed to Whisnant, A. W..

7 recordsLinked to original sources

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↗

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↗

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

Herpes simplex virus 1 inhibits phosphorylation of RNA polymerase II CTD serine-7

Transcriptional activity of RNA polymerase II (Pol II) is orchestrated by post-translational modifications of the C-terminal domain (CTD) of the largest Pol II subunit, RPB1. Herpes Simplex Virus type 1 (HSV-1) usurps the cellular transcriptional machinery during lytic infection to efficiently express viral mRNA and shut down host gene expression. The viral immediate-early protein ICP22 interferes with serine 2 phosphorylation (pS2) of the Pol II CTD by targeting CDK9. The functional implications of this are poorly understood. Here, we report that HSV-1 also induces a global loss of serine 7 phosphorylation (pS7). This effect was dependent on the expression of the two viral immediate-early proteins, ICP22 and ICP27. While lytic HSV-1 infection results in efficient Pol II degradation late in infection, we show that pS2/S7 loss precedes the drop in Pol II level. Interestingly, mutation of the RPB1 polyubiquitination site mutation K1268, which prevents proteasomal RPB1 degradation during transcription-coupled DNA repair, displayed loss of pS2/S7 but retained much higher overall RPB1 protein levels even at late times of infection, indicating that this pathway mediates bulk Pol II protein loss late in infection but is not involved in early CTD dysregulation. Using -amanitin-resistant CTD mutants, we observed differential requirements for Ser2 and Ser7 for production of viral proteins, with Ser2 facilitating viral immediate-early gene expression and Ser7 appearing dispensable. Despite dysregulation of CTD phosphorylation and different requirements for Ser2/7, all CTD modifications tested could be visualized in viral replication compartments by immunofluorescence. These data expand the known means that HSV-1 employs to create pro-viral transcriptional environments at the expense of host responses.

molecular biology↗

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

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↗