Biology subjects

Depledge, D. P.

Publications and source records attributed to Depledge, D. P..

16 recordsLinked to original sources

bioRxiv2026

An ONT sequencing framework for hantavirus genome assembly, variant analysis, and defective viral genome characterization from whole-segment amplicons

Orthohantaviruses are zoonotic, tri-segmented negative-sense RNA viruses of rodents and other small mammals that can cause severe human disease. Whole genome sequencing remains difficult because of low viral loads, high sequence diversity, and an inherent instability of the viral L segment. Current tiled amplicon and hybrid-capture approaches do not capture the heterogeneity of virus populations, including defective viral genomes (DVGs), which have never been described for hantaviruses. Here, we present an Oxford Nanopore Technologies sequencing framework based on universal reverse transcription primers targeting the conserved genome termini of orthohantaviruses and a single species-specific primer pair per segment, enabling amplification and sequencing of complete S, M, and L segments. We also developed an accompanying bioinformatic pipeline that performs reference-guided assembly of hantavirus genomes, consensus generation, variant calling, and DVG classification using the newly developed ORCHID classification pipeline. We applied this approach to diverse Puumala virus strains from infected cell cultures and bank voles, to representative Old World and New World orthohantaviruses, and to highly pathogenic New World viruses in vivo. In all cases we were able to accurately reconstruct full hantavirus genomes. Notably, we found that hantavirus populations contain highly abundant DVGs and conserved L segment deletions, whereas the S and M segments remained largely intact. Together, this work provides a low-cost, broadly applicable and easily customisable workflow for hantavirus genomics and reveals DVGs as a previously unrecognized feature of hantavirus populations.

microbiology↗

bioRxiv2025

Extended poly(A) tails are a shared feature of herpesvirus mRNAs

Poly(A) tails are present on most cellular and viral mRNAs, providing a platform for poly(A)-binding proteins that stimulate translation and regulate the deadenylation and stability of transcripts in the cytoplasm. Here we leverage nanopore direct RNA sequencing to analyse the distribution of poly(A) tail lengths on cellular and viral mRNAs across Herpesviridae and other DNA and RNA virus infections. We find that herpesvirus mRNA poly(A) tails are consistently longer than those on cellular and other viral transcripts, presenting a previously unrecognized yet widespread mechanism to advantage herpesviral gene expression. This contrasts with the templated poly(A) tails on coronavirus RNAs and those on cytoplasmically transcribed poxviral mRNAs, which are more similar in length to those on host mRNAs. Herpesviral noncoding RNAs display differential poly(A) tailing patterns which do not correlate with nuclear localisation while individual herpesviral mRNAs also show variation in the extent to which their poly(A) tail lengths change during the virus lifecycle, suggestive of additional uncharacterised layers of poly(A) tail length regulation. Importantly, while we detect non-adenosine nucleotides within herpesviral poly(A) tails, which are known to oppose deadenylase activity, this "mixed tailing" is not at sufficient frequency to explain the widespread extended tails of herpesvirus mRNAs.

microbiology↗

bioRxiv2025

Establishing benchmarks for quantitative mapping of m6A by Nanopore Direct RNA Sequencing

Nanopore direct RNA sequencing (DRS) coupled with Dorado modification-aware basecalling enables mapping of epitranscriptomic modifications including N6-methyladenosine (m6A) at the level of individual RNAs. However, the sensitivity, specificity, and reproducibility of this method remain unclear and have only recently begun to be addressed through systematic benchmarking studies. Here, we aimed to establish a best-practice workflow for DRS-based epitranscriptomic analyses. Specifically, we evaluated multiple Dorado versions and models using RNA isolated from primary cells and unmodified in vitro transcribed RNAs. We further utilized an m6A methyltransferase inhibitor as a specificity control. We established that stringent filtering is necessary to reduce false-positive calls and found that Dorado predictions captured an increasing proportion of GLORI sites detected at high m6A/A proportions. Further, by applying DRS to human primary fibroblasts and HD10.6 neurons, we detected cell type-specific differences in the predicted m6A/A proportions at conserved sites. Our study thus presents the first systematic comparison of Dorado and GLORI from the same input RNA and expands characterization of the m6A epitranscriptome to fibroblasts and neurons.

bioinformatics↗

bioRxiv2025

Defining expansions and perturbations to the RNA polymerase III transcriptome and epitranscriptome by modified direct RNA nanopore sequencing

RNA polymerase III (Pol III) transcribes cytosolic transfer RNAs (tRNAs) and other non-coding RNAs (ncRNAs) essential to cellular function. However, many aspects of Pol III transcription and processing, including RNA modifications, remain poorly understood, mainly due to a lack of available sensitive and systematic methods for their analysis. Here, we present DRAP3R (Direct Read and Analysis of Polymerase III transcribed RNAs), a modified nanopore direct RNA sequencing approach and analysis framework that enables the specific and sensitive capture of nascent Pol III transcribed RNAs. Applying DRAP3R to distinct cell types, we identify previously unconfirmed tRNA genes and other novel Pol III transcribed RNAs, thus expanding the known Pol III transcriptome. Critically, DRAP3R also enables discrimination between co- and post-transcriptional RNA modifications such as pseudouridine ({Psi}) and N6-methyladenosine (m6A) at single-nucleotide resolution across all examined transcript types and reveals differential {Psi} installation patterns across tRNA isodecoders and other ncRNAs. Finally, applying DRAP3R to epithelial cells infected with Herpes Simplex Virus Type 1 reveals an extensive remodelling of both the Pol III transcriptome and epitranscriptome. Our findings thus establish DRAP3R as a powerful tool for systematically studying Pol III transcribed RNAs and their modifications in diverse cellular contexts.

systems biology↗

bioRxiv2024

Repression of varicella zoster virus gene expression during quiescent infection in the absence of detectable histone deposition

Varicella zoster virus (VZV) is a human-specific herpesvirus that establishes latency in peripheral neurons. The only transcripts detected in infected human trigeminal ganglia (TG) obtained shortly after death correspond to the VZV latency-associated transcript (VLT) and associated VLT-ORF63 splice variants. In vitro studies showed that VLT-ORF63 is translated into a protein (pVLT-ORF63) that induces VZV transcription. The mechanisms that lead to this restricted gene expression and the transition to lytic replication remain unknown, partly due to the difficulty of working with human neurons. In this study, we addressed whether the neuroblastoma-derived cell line SH-SY5Y could serve as a model to investigate the mechanisms that lead to repression of VZV gene expression followed by reactivation. VZV productively infected differentiated SH-SY5Y (dSH-SY5Y) whereas incubation with acyclovir (ACV) inhibited virus replication and induced a progressive repression of the virus. Upon removal of ACV there was production of viral particles in a subset of cells, while others contained non-replicating VZV genomes and VLT-containing transcripts for at least 20 days post-infection (dpi). Exogenous expression of VLT-ORF63 induced productive infection, suggesting that the non-replicating and repressed genomes remained functional. Interestingly, histone deposition was undetectable at VZV genomes in quiescently infected dSH-SY5Y cells, pointing to a potential novel mechanism leading to VZV repression in this neuronal setting.

microbiology↗

bioRxiv2024

Nanopore Guided Annotation of Transcriptome Architectures

High-resolution annotations of transcriptomes from all domains of life are essential for many sequencing-based RNA analyses, including Nanopore direct RNA sequencing (DRS), which would otherwise be hindered by misalignments and other analysis artefacts. DRS allows the capture and full-length sequencing of native RNAs, without recoding or amplification bias, and resulting data may be interrogated to define the identity and location of chemically modified ribonucleotides, as well as the length of poly(A) tails on individual RNA molecules. Existing software solutions for generating high-resolution transcriptome annotations are poorly suited to small gene dense organisms such as viruses due to the challenge of identifying distinct transcript isoforms where alternative splicing and overlapping RNAs are prevalent. To resolve this, we identified key characteristics of DRS datasets and developed a novel approach to transcriptome. We demonstrate, using a combination of synthetic and original datasets, that our novel approach yields a high level of precision and recall when reconstructing both gene sparse and gene dense transcriptomes from DRS datasets. We further apply this approach to generate a new high resolution transcriptome annotation of the neglected pathogen human adenovirus type F 41 for which we identify 77 distinct transcripts encoding at least 23 different proteins.

bioinformatics↗

bioRxiv2023

Histone H2A variant H2A.B is enriched in transcriptionally active HSV-1 lytic chromatin

Herpes simplex virus 1 (HSV-1) transcription is restricted in latently infected neurons and the genomes are in mostly silenced chromatin, whereas all viral genes are transcribed in lytically infected cells, in which the genomes are dynamically chromatinized. Epigenetic regulation modulates HSV-1 transcription during lytic, latent, and reactivating infections, but the precise mechanisms are not fully defined. Nucleosomes are dynamic; they slide, breathe, assemble and disassemble. We and others have proposed that the most dynamic HSV-1 chromatin is transcriptionally competent whereas the least dynamic is silenced. However, the mechanisms yielding the unusually dynamic viral chromatin remain unknown. Histone variants affect nucleosome dynamics. The dynamics of H2A, H2A.X and macroH2A were enhanced in infected cells, whereas those of H2A.B uniquely decreased. We constructed stably transduced cells expressing tagged histone H2A, H2A.B, macroH2A, or H2B, which assembles the H2A/H2B nucleosome dimers with all H2A variants. All H2A variants, ectopic, and endogenous H2B, were assembled into HSV-1 chromatin evenly throughout the genome, but canonical H2A was relatively depleted from the viral chromatin whereas H2A.B was enriched in the most dynamic viral chromatin. When viral transcription was restricted, H2A.B became as depleted from the viral chromatin through the entire genome as H2A. We propose that lytic HSV-1 nucleosomes are enriched in the dynamic variant H2A.B/H2B dimers to promote HSV-1 chromatin dynamics and transcriptional competency, and conclude that the dynamics of HSV-1 chromatin are determined in part by the H2A variants. ImportanceHSV-1 transcription is epigenetically regulated during latent and lytic infections, and epigenetic inhibitors have been proposed as potential antiviral drugs to modulate latency and reactivation. However, the detailed mechanisms of regulation of HSV-1 transcription by epigenetics have not been fully characterized and may differ from those regulating cellular transcription. In particular, the lytic HSV-1 chromatin is unusually dynamic, whereas the latent silenced one is not, but the mechanisms resulting in the unique dynamics of the lytic chromatin remain unknown. Here we identify the enrichment on the highly dynamic histone 2A variant H2A in the most dynamic viral chromatin, which provides a mechanistic understanding for its unique dynamics. Future work to identify the mechanisms of enrichment in H2A.B on the viral chromatin may identify novel druggable epigenetic regulators that modulate HSV-1 latency and reactivation.

microbiology↗

bioRxiv2023

Reanalysis of single-cell RNA sequencing data does not support herpes simplex virus 1 latency in non-neuronal ganglionic cells in mice

Most individuals are latently infected with herpes simplex virus type 1 (HSV-1) and it is well-established that HSV-1 establishes latency in sensory neurons of peripheral ganglia. However, it was recently proposed that latent virus is also present in immune cells recovered from ganglia in a mouse model used for studying latency. Here, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data that formed the basis for this conclusion. Unexpectedly, off-target priming in 3 scRNA-Seq experiments enabled the detection of non-polyadenylated HSV-1 latency-associated transcript (LAT) intronic RNAs. However, LAT reads were near-exclusively detected in a mixed population of cells undergoing cell death. Specific loss of HSV-1 LAT and neuronal transcripts during quality control filtering indicated widespread destruction of neurons, supporting the presence of contaminating cell-free RNA in other cells following tissue processing. In conclusion, the reported detection of latent HSV-1 in non-neuronal cells is best explained by inaccuracies in the data analyses.

microbiology↗

bioRxiv2022

DLK-dependent biphasic reactivation of herpes simplex virus latency established in the absence of antivirals

Understanding the molecular mechanism of herpes simplex virus type 1 (HSV-1) latent infection and reactivation in neurons requires the use of model systems. However, establishing a quiescent infection in cultured neurons is problematic as infectious virus released from any productively infected neuron can superinfect the cultures. Here, we describe a new reporter virus, HSV-1 Stayput-GFP, that is defective for cell-to-cell spread and can be used to model latency and reactivation at the single neuron level. Importantly, quiescent infection of neurons with Stayput-GFP can be established without the use of a viral DNA replication inhibitor. The establishment of a quiescent state requires a longer time frame than previous models of HSV latency using DNA replication inhibitors. This results in a decreased ability of the virus to reactivate, and the use of multiple reactivation triggers is required. Using this system, we demonstrate that an initial Phase I wave of lytic gene expression occurs independently of histone demethylase activity and viral DNA replication but is dependent on the neuronal cell stress protein, DLK. Progression into the later, Phase II wave of reactivation, characterized by detectable late viral protein synthesis and a requirement for histone demethylase activity, is also observed with the Stayput-GFP model system. These data demonstrate that the two waves of viral gene expression following HSV-1 reactivation are independent of secondary infection and occur when latent infections are established in the absence of a viral DNA replication inhibitor. ImportanceHerpes simplex virus-1 (HSV-1) enters a latent infection in neurons and periodically reactivates. Reactivation manifests as a variety of clinical symptoms. Studying latency and reactivation in vitro is invaluable, allowing the molecular mechanisms behind both processes to be targeted by therapeutics that reduce the clinical consequences. Here, we describe a novel in vitro model system using a cell-to-cell spread defective HSV-1, known as Stayput-GFP, which allows for the study of latency and reactivation at the single neuron level. We anticipate this new model system will be an incredibly valuable tool for studying the establishment and reactivation of HSV-1 latent infection in vitro. Using this model, we find that initial reactivation events are dependent on cellular stress kinase DLK, but independent of histone demethylase activity and viral DNA replication. Our data therefore demonstrate the essential role of DLK in mediating a wave of lytic gene expression unique to reactivation.

microbiology↗

bioRxiv2020

Expression of varicella-zoster virus VLT-ORF63 fusion transcript induces broad viral gene expression during reactivation from neuronal latency

Varicella-zoster virus (VZV) establishes lifelong neuronal latency in most humans world-wide, reactivating in one-third to cause herpes zoster and occasionally chronic pain. How VZV establishes, maintains and reactivates from latency is largely unknown. Latent VZV gene expression is restricted to VZV latency-associated transcript (VLT) and open reading frame 63 (ORF63) in naturally VZV-infected human trigeminal ganglia (TG). Notably, these transcript levels positively correlated suggesting co-regulated transcription during latency. Here, we used direct RNA-sequencing to identify fusion transcripts that combine VLT and ORF63 loci (VLT-ORF63) and are expressed during both lytic and latent VZV infections. Furthermore, real-time PCR, RNA in situ hybridization and 5 rapid amplification of cDNA ends (RACE) all confirmed VLT-ORF63, but not canonical ORF63, expression in human TG. During lytic infection, one of the two major VLT-ORF63 isoforms encodes a novel fusion protein combining VLT and ORF63 proteins (pVLT-ORF63). In vitro, VLT is transcribed in latently VZV-infected human sensory neurons, whereas VLT-ORF63 expression is induced by reactivation stimuli. Moreover, the pVLT-ORF63-encoding VLT-ORF63 isoform induced transcription of lytic VZV genes. Collectively, our findings show that VZV expresses a unique set of VLT-ORF63 transcripts, potentially involved in the transition from latency to lytic VZV infection.

microbiology↗

bioRxiv2020

Decoding the architecture of the varicella-zoster virus transcriptome

Varicella-zoster virus (VZV), a double-stranded DNA virus, causes varicella, establishes lifelong latency in ganglionic neurons, and reactivates later in life to cause herpes zoster, commonly associated with chronic pain. The VZV genome is densely packed and produces multitudes of overlapping transcripts deriving from both strands. While 71 distinct open reading frames (ORFs) have thus far been experimentally defined, the full coding potential of VZV remains unknown. Here, we integrated multiple short-read RNA sequencing approaches with long-read direct RNA sequencing on RNA isolated from VZV-infected cells to provide a comprehensive reannotation of the lytic VZV transcriptome architecture. Through precise mapping of transcription start sites, splice junctions, and polyadenylation sites, we identified 136 distinct polyadenylated VZV RNAs that encode canonical ORFs, non-canonical ORFs, and ORF fusions, as well as putative non-coding RNAs (ncRNAs). Furthermore, we determined the kinetic class of all VZV transcripts and observed, unexpectedly, that transcripts encoding the ORF62 protein, previously designated as immediate-early, were expressed with late kinetics. Our work showcases the complexity of the VZV transcriptome and provides a comprehensive resource that will facilitate future functional studies of coding RNAs, ncRNAs, and the biological mechanisms underlying the regulation of viral transcription and translation during lytic VZV infection.

microbiology↗

bioRxiv2019

Novel splicing and open reading frames revealed by long-read direct RNA sequencing of adenovirus transcripts

Adenovirus is a common human pathogen that relies on host cell processes for production and processing of viral RNA. Although adenoviral promoters, splice junctions, and cleavage and polyadenylation sites have been characterized using low-throughput biochemical techniques or short read cDNA-based sequencing, these technologies do not fully capture the complexity of the adenoviral transcriptome. By combining Illumina short-read and nanopore long-read direct RNA sequencing approaches, we mapped transcription start sites and cleavage and polyadenylation sites across the adenovirus genome. The canonical viral early and late RNA cassettes were confirmed, but analysis of splice junctions within long RNA reads revealed an additional 20 novel viral transcripts. These RNAs include seven new splice junctions which lead to expression of canonical open reading frames (ORF), as well as 13 transcripts encoding for messages that potentially alter protein functions through truncations or the fusion of canonical ORFs. In addition, we also detect RNAs that bypass canonical cleavage sites and generate potential chimeric proteins by linking separate gene transcription units. Our work highlights how long-read sequencing technologies can reveal further complexity within viral transcriptomes.

microbiology↗

bioRxiv2019

Whole genome sequencing of Herpes Simplex Virus 1 directly from human cerebrospinal fluid reveals selective constraints in neurotropic viruses.

Herpes Simplex Virus type 1 (HSV-1) chronically infects over 70% of the global population. Clinical manifestations are largely restricted to recurrent epidermal vesicles. However, HSV-1 also leads to encephalitis, the infection of the brain parenchyma, with high associated rates of mortality and morbidity. In this study, we performed target enrichment followed by direct sequencing of HSV-1 genomes, using target enrichment methods on the cerebrospinal fluid (CSF) of clinical encephalitis patients and from skin swabs of epidermal vesicles on non-encephalopathic patients. Phylogenetic analysis revealed high inter-host diversity and little population structure. By contrast, samples from different lesions in the same patient clustered with similar patterns of allelic variants. Comparison of consensus genome sequences shows HSV-1 has been freely recombining, except for distinct islands of linkage disequilibrium (LD). This suggests functional constraints prevent recombination between certain genes, notably those encoding pairs of interacting proteins. Distinct LD patterns characterised subsets of viruses recovered from CSF and skin lesions, which may reflect different evolutionary constraints in different body compartments. Functions of genes under differential constraint related to immunity or tropism and provide new hypotheses on tissue-specific mechanisms of viral infection and latency.

evolutionary biology↗

bioRxiv2019

Direct RNA sequencing reveals m6A modifications on adenovirus RNA are necessary for efficient splicing

Adenovirus is a nuclear replicating DNA virus reliant on host RNA processing machinery. Processing and metabolism of cellular RNAs can be regulated by METTL3, which catalyzes the addition of N6-methyladenosine (m6A) to mRNAs. While m6A-modified adenoviral RNAs have been previously detected, the location and function of this mark within the infectious cycle is unknown. Since the complex adenovirus transcriptome includes overlapping spliced units that would impede accurate m6A mapping using short-read sequencing, we profiled m6A within the adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read sequencing to yield both nucleotide and transcript-resolved m6A detection. Although both early and late viral transcripts contain m6A, depletion of m6A writer METTL3 specifically impacts viral late transcripts by reducing their splicing efficiency. These data showcase a new technique for m6A discovery within individual transcripts at nucleotide resolution, and highlight the role of m6A in regulating splicing of a viral pathogen.

molecular biology↗

bioRxiv2018

Native RNA sequencing on nanopore arrays redefines the transcriptional complexity of a viral pathogen

Viral genomes exhibit a higher gene density and more diversified transcriptome than the host cell. Coding potential is maximized through the use of multiple reading frames, placement of genes on opposing strands, inefficient or modified use of termination signals, and the deployment of complex alternative splicing patterns. As a consequence, detailed characterization of viral transcriptomes by conventional methods can be challenging. Full length native RNA sequencing (nRNA-seq) using nanopore arrays offers an exciting alternative. Individual transcripts are sequenced directly, without the biases inherent to the recoding or amplification steps included in other sequencing methodologies. nRNA-seq simplifies the detection of variation brought about by RNA splicing, use of alternative transcription initiation and termination sites, and other RNA modifications. Here we use nRNA-seq to profile the herpes simplex virus type 1 transcriptome during early and late stages of productive infection of primary cells. We demonstrate the effectiveness of the approach and identify a novel class of intergenic transcripts, including an mRNA that accumulates late in infection that codes for a novel fusion of the viral E3 ubiquitin ligase ICP0 and viral membrane glycoprotein L.

microbiology↗

bioRxiv2017

Newly identified varicella-zoster virus latency transcript inhibits viral replication

During primary infection, neurotropic alphaherpesviruses (HVs) gain access to neurons in sensory and cranial ganglia establishing lifelong latent infection from which they can later reactivate to cause debilitating disease1. For most HVs, including the best-studied herpes simplex type 1 ( HSV-1), viral latency is characterized by expression of a single or restricted set of transcripts that map antisense to the open reading frame (ORF) homologous to the major HSV immediate early viral transactivator, ICP02. These latency transcripts, either directly or through encoded miRNAs or proteins, repress expression of the ICP0 orthologues3-5. The exception is varicella-zoster virus (VZV), an HV which infects over 90% of adults and for which neither a canonical latency transcript1,6-8 nor a putative mechanism for repressing lytic transcription during latency have been identified. Here, we describe the discovery and functional characterization of a VZV latency transcript (VLT), that maps antisense to VZV ORF 61 (the VZV ICP0 homologue9,10), and which is consistently expressed in neurons of latently infected human trigeminal ganglia (TG). VLT encodes a protein with late kinetics during lytic VZV infection in vitro and in zoster skin lesions. Whereas multiple alternatively spliced VLT isoforms are expressed during lytic VZV infection, a single unique VLT isoform that specifically suppresses ORF61 gene expression predominates in latently VZV-infected human TG. The discovery of VLT directly unifies the latent VZV transcription program with those of better-characterized HVs, removing longstanding barriers to understanding VZV latency and paving the way for research into the development of vaccines that do not establish latency or reactivate, and drugs that eradicate latent VZV.

microbiology↗