RESUMO
Nanopore direct RNA sequencing (DRS) enables the capture and full-length sequencing of native RNAs, without recoding or amplification bias. Resulting data sets 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. The success of these analyses is highly dependent on the provision of high-resolution transcriptome annotations in combination with workflows that minimize misalignments and other analysis artifacts. Existing software solutions for generating high-resolution transcriptome annotations are poorly suited to small gene-dense genomes of 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 data sets that inform resulting read alignments and developed the nanopore guided annotation of transcriptome architectures (NAGATA) software package (https://github.com/DepledgeLab/NAGATA). We demonstrate, using a combination of synthetic and original DRS data sets derived from adenoviruses, herpesviruses, coronaviruses, and human cells, that NAGATA outperforms existing transcriptome annotation software and yields a consistently high level of precision and recall when reconstructing both gene sparse and gene-dense transcriptomes. Finally, we apply NAGATA to generate the first high-resolution transcriptome annotation of the neglected pathogen human adenovirus type F41 (HAdV-41) for which we identify 77 distinct transcripts encoding at least 23 different proteins. IMPORTANCE: The transcriptome of an organism denotes the full repertoire of encoded RNAs that may be expressed. This is critical to understanding the biology of an organism and for accurate transcriptomic and epitranscriptomic-based analyses. Annotating transcriptomes remains a complex task, particularly in small gene-dense organisms such as viruses which maximize their coding capacity through overlapping RNAs. To resolve this, we have developed a new software nanopore guided annotation of transcriptome architectures (NAGATA) which utilizes nanopore direct RNA sequencing (DRS) datasets to rapidly produce high-resolution transcriptome annotations for diverse viruses and other organisms.
Assuntos
Anotação de Sequência Molecular , Software , Transcriptoma , Humanos , Transcriptoma/genética , Anotação de Sequência Molecular/métodos , Análise de Sequência de RNA/métodos , Herpesviridae/genética , Coronavirus/genética , Sequenciamento por Nanoporos/métodos , Nanoporos , Adenoviridae/genéticaRESUMO
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.
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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 HSV-1 is also present in immune cells recovered from the ganglia of experimentally infected mice. Here, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data that formed the basis for that 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 mixed populations 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 using compromised scRNA-Seq datasets.IMPORTANCEMost people are infected with herpes simplex virus type 1 (HSV-1) during their life. Once infected, the virus generally remains in a latent (silent) state, hiding within the neurons of peripheral ganglia. Periodic reactivation (reawakening) of the virus may cause fresh diseases such as cold sores. A recent study using single-cell RNA sequencing (scRNA-Seq) proposed that HSV-1 can also establish latency in the immune cells of mice, challenging existing dogma. We reanalyzed the data from that study and identified several flaws in the methodologies and analyses performed that invalidate the published conclusions. Specifically, we showed that the methodologies used resulted in widespread destruction of neurons which resulted in the presence of contaminants that confound the data analysis. We thus conclude that there remains little to no evidence for HSV-1 latency in immune cells.
Assuntos
Artefatos , Gânglios Sensitivos , Herpesvirus Humano 1 , Células Receptoras Sensoriais , Análise de Sequência de RNA , Análise da Expressão Gênica de Célula Única , Latência Viral , Animais , Camundongos , Morte Celular , Conjuntos de Dados como Assunto , Gânglios Sensitivos/imunologia , Gânglios Sensitivos/patologia , Gânglios Sensitivos/virologia , Herpes Simples/imunologia , Herpes Simples/patologia , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/isolamento & purificação , MicroRNAs/análise , MicroRNAs/genética , Reprodutibilidade dos Testes , RNA Viral/análise , RNA Viral/genética , Células Receptoras Sensoriais/patologia , Células Receptoras Sensoriais/virologiaRESUMO
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 nearexclusively detected in a mixed population of cells undergoing cell death. Specific loss of HSV1 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.
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Herpes simplex virus type-1 (HSV-1) protein ICP27 is an essential immediate early (IE) protein that promotes the expression of viral early (E) and late (L) genes via multiple mechanisms. Our understanding of this complex regulatory protein has been greatly enhanced by the characterization of HSV-1 mutants bearing engineered alterations in the ICP27 gene. However, much of this analysis has been performed in interferon-deficient Vero monkey cells. Here, we assessed the replication of a panel of ICP27 mutants in several other cell types. Our analysis shows that mutants lacking ICP27's amino (N)-terminal nuclear export signal (NES) display a striking cell type-dependent growth phenotype, i.e., they grow semi-permissively in Vero and some other cells but are tightly blocked for replication in primary human fibroblasts and multiple human cell lines. This tight growth defect correlates with a failure of these mutants to replicate viral DNA. We also report that HSV-1 NES mutants are deficient in expressing the IE protein ICP4 at early times postinfection. Analysis of viral RNA levels suggests that this phenotype is due, at least in part, to a defect in the export of ICP4 mRNA to the cytoplasm. In combination, our results (i) show that ICP27's NES is critically important for HSV-1 replication in many human cells, and (ii) suggest that ICP27 plays a heretofore unappreciated role in the expression of ICP4. IMPORTANCE HSV-1 IE proteins drive productive HSV-1 replication. The major paradigm of IE gene induction, developed over many years, involves the parallel activation of the five IE genes by the viral tegument protein VP16, which recruits the host RNA polymerase II (RNAP II) to the IE gene promoters. Here, we provide evidence that ICP27 can enhance ICP4 expression early in infection. Because ICP4 is required for transcription of viral E and L genes, this finding may be relevant to understanding how HSV-1 enters and exits the latent state in neurons.
Assuntos
Herpesvirus Humano 1 , Proteínas Imediatamente Precoces , Animais , Chlorocebus aethiops , Humanos , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Sinais de Exportação Nuclear , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Linhagem Celular , Células Vero , Replicação ViralRESUMO
Adenovirus is a common human pathogen that relies on host cell processes for transcription and processing of viral RNA and protein production. Although adenoviral promoters, splice junctions, 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 RNA cleavage and polyadenylation sites across the adenovirus genome. In addition to confirming the known canonical viral early and late RNA cassettes, our analysis of splice junctions within long RNA reads revealed an additional 35 novel viral transcripts that meet stringent criteria for expression. These RNAs include fourteen new splice junctions which lead to expression of canonical open reading frames (ORFs), six novel ORF-containing transcripts, and 15 transcripts encoding for messages that could alter protein functions through truncation or fusion of canonical ORFs. In addition, we detect RNAs that bypass canonical cleavage sites and generate potential chimeric proteins by linking distinct gene transcription units. Among these chimeric proteins we detected an evolutionarily conserved protein containing the N-terminus of E4orf6 fused to the downstream DBP/E2A ORF. Loss of this novel protein, E4orf6/DBP, was associated with aberrant viral replication center morphology and poor viral spread. Our work highlights how long-read sequencing technologies combined with mass spectrometry can reveal further complexity within viral transcriptomes and resulting proteomes.
Assuntos
Adenoviridae , RNA Viral , Adenoviridae/genética , DNA Complementar , Humanos , Fases de Leitura Aberta/genética , Proteoma/metabolismo , Splicing de RNA/genética , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de RNA/métodos , TranscriptomaRESUMO
Methylation at the N6-position of either adenosine (m6A) or 2'-O-methyladenosine (m6Am) represents two of the most abundant internal modifications of coding and non-coding RNAs, influencing their maturation, stability and function. Additionally, although less abundant and less well-studied, monomethylation at the N1-position (m1A) can have profound effects on RNA folding. It has been known for several decades that RNAs produced by both DNA and RNA viruses can be m6A/m6Am modified and the list continues to broaden through advances in detection technologies and identification of the relevant methyltransferases. Recent studies have uncovered varied mechanisms used by viruses to manipulate the m6A pathway in particular, either to enhance virus replication or to antagonize host antiviral defenses. As such, RNA modifications represent an important frontier of exploration in the broader realm of virus-host interactions, and this new knowledge already suggests exciting opportunities for therapeutic intervention. In this review we summarize the principal mechanisms by which m6A/m6Am can promote or hinder viral replication, describe how the pathway is actively manipulated by biomedically important viruses, and highlight some remaining gaps in understanding how adenosine methylation of RNA controls viral replication and pathogenesis.
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Adenosina , Vírus , Adenosina/genética , Adenosina/metabolismo , Animais , Metilação , RNA/genética , RNA/metabolismo , Replicação Viral , Vírus/genéticaRESUMO
A signature trait of neurotropic α-herpesviruses (α-HV) is their ability to establish stable non-productive infections of peripheral neurons termed latency. This specialized gene expression program is the foundation of an evolutionarily successful strategy to ensure lifelong persistence in the host. Various physiological stresses can induce reactivation in a subset of latently-infected neurons allowing a new cycle of viral productive cycle gene expression and synthesis of infectious virus. Recurring reactivation events ensure transmission of the virus to new hosts and contributes to pathogenesis. Efforts to define the molecular basis of α-HV latency and reactivation have been notoriously difficult because the neurons harboring latent virus in humans and in experimentally infected live-animal models, are rare and largely inaccessible to study. Increasingly, researchers are turning to cultured neuron infection models as simpler experimental platforms from which to explore latency and reactivation at the molecular level. In this review, I reflect on the strengths and weaknesses of existing neuronal models and briefly summarize the important mechanistic insights these models have provided. I also discuss areas where prioritization will help to ensure continued progress and integration.
Assuntos
Herpesviridae , Herpesvirus Humano 1 , Animais , Herpesvirus Humano 1/fisiologia , Neurônios , Vírus Oncogênicos , Ativação Viral/fisiologia , Latência Viral/fisiologiaRESUMO
Understanding the molecular mechanisms of herpes simplex virus 1 (HSV-1) latent infection and reactivation in neurons requires the use of in vitro model systems. Establishing a quiescent infection in cultured neurons is problematic, as any infectious virus released can superinfect the cultures. Previous studies have used the viral DNA replication inhibitor acyclovir to prevent superinfection and promote latency establishment. Data from these previous models have shown that reactivation is biphasic, with an initial phase I expression of all classes of lytic genes, which occurs independently of histone demethylase activity and viral DNA replication but is dependent on the cell stress protein DLK. Here, we describe a new model system using HSV-1 Stayput-GFP, a reporter virus that is defective for cell-to-cell spread and establishes latent infections without the need for acyclovir. The establishment of a latent state requires a longer time frame than previous models using DNA replication inhibitors. This results in a decreased ability of the virus to reactivate using established inducers, and as such, a combination of reactivation triggers is required. Using this system, we demonstrate that biphasic reactivation occurs even when latency is established in the absence of acyclovir. Importantly, phase I lytic gene expression still occurs in a histone demethylase and viral DNA replication-independent manner and requires DLK activity. These data demonstrate that the two waves of viral gene expression following HSV-1 reactivation are independent of secondary infection and not unique to systems that require acyclovir to promote latency establishment. IMPORTANCE Herpes 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 further validate the essential role of DLK in mediating a wave of lytic gene expression unique to reactivation.
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Herpes Simples , Herpesvirus Humano 1 , Infecção Latente , MAP Quinase Quinase Quinases , Ativação Viral , Latência Viral , Aciclovir/farmacologia , Antivirais/farmacologia , Replicação do DNA , DNA Viral , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Histona Desmetilases/genética , Humanos , MAP Quinase Quinase Quinases/metabolismo , Replicação ViralRESUMO
MOTIVATION: The chemical modification of ribonucleotides regulates the structure, stability and interactions of RNAs. Profiling of these modifications using short-read (Illumina) sequencing techniques provides high sensitivity but low-to-medium resolution i.e. modifications cannot be assigned to specific transcript isoforms in regions of sequence overlap. An alternative strategy uses current fluctuations in nanopore-based long read direct RNA sequencing (DRS) to infer the location and identity of nucleotides that differ between two experimental conditions. While highly sensitive, these signal-level analyses require high-quality transcriptome annotations and thus are best suited to the study of model organisms. By contrast, the detection of RNA modifications in microbial organisms which typically have no or low-quality annotations requires an alternative strategy. Here, we demonstrate that signal fluctuations directly influence error rates during base-calling and thus provides an alternative approach for identifying modified nucleotides. RESULTS: DRUMMER (Detection of Ribonucleic acid Modifications Manifested in Error Rates) (i) utilizes a range of statistical tests and background noise correction to identify modified nucleotides with high confidence, (ii) operates with similar sensitivity to signal-level analysis approaches and (iii) correlates very well with orthogonal approaches. Using well-characterized DRS datasets supported by independent meRIP-Seq and miCLIP-Seq datasets we demonstrate that DRUMMER operates with high sensitivity and specificity. AVAILABILITY AND IMPLEMENTATION: DRUMMER is written in Python 3 and is available as open source in the GitHub repository: https://github.com/DepledgeLab/DRUMMER. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Sequenciamento por Nanoporos , Software , Análise de Sequência de RNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/química , NucleotídeosRESUMO
Single-cell RNA sequencing (scRNA-seq) is a powerful technique for dissecting the complexity of normal and diseased tissues, enabling characterization of cell diversity and heterogeneous phenotypic states in unprecedented detail. However, this technology has been underutilized for exploring the interactions between the host cell and viral pathogens in latently infected cells. Herein, we use scRNA-seq and single-molecule sensitivity fluorescent in situ hybridization (smFISH) technologies to investigate host single-cell transcriptome changes upon the reactivation of a human neurotropic virus, herpes simplex virus-1 (HSV-1). We identify the stress sensor growth arrest and DNA damage-inducible 45 beta (Gadd45b) as a critical antiviral host factor that regulates HSV-1 reactivation events in a subpopulation of latently infected primary neurons. We show that distinct subcellular localization of Gadd45b correlates with the viral late gene expression program, as well as the expression of the viral transcription factor, ICP4. We propose that a hallmark of a "successful" or "aborted" HSV-1 reactivation state in primary neurons is determined by a unique subcellular localization signature of the stress sensor Gadd45b.
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Antígenos de Diferenciação/metabolismo , Herpesvirus Humano 1 , Neurônios/virologia , Ativação Viral , Latência Viral , Regulação da Expressão Gênica , Herpesvirus Humano 1/fisiologia , Humanos , Hibridização in Situ Fluorescente , TranscriptomaRESUMO
N6-methyladenosine (m6A) is the most abundant internal messenger RNA (mRNA) modification, contributing to the processing, stability, and function of methylated RNAs. Methylation occurs in the nucleus during pre-mRNA synthesis and requires a core methyltransferase complex consisting of METTL3, METTL14, and WTAP. During herpes simplex virus (HSV-1) infection, cellular gene expression is profoundly suppressed, allowing the virus to monopolize the host transcription and translation apparatus and antagonize antiviral responses. The extent to which HSV-1 uses or manipulates the m6A pathway is not known. Here, we show that, in primary fibroblasts, HSV-1 orchestrates a striking redistribution of the nuclear m6A machinery that progresses through the infection cycle. METTL3 and METTL14 are dispersed into the cytoplasm, whereas WTAP remains nuclear. Other regulatory subunits of the methyltransferase complex, along with the nuclear m6A-modified RNA binding protein YTHDC1 and nuclear demethylase ALKBH5, are similarly redistributed. These changes require ICP27, a viral regulator of host mRNA processing that mediates the nucleocytoplasmic export of viral late mRNAs. Viral gene expression is initially reduced by small interfering RNA (siRNA)-mediated inactivation of the m6A methyltransferase but becomes less impacted as the infection advances. Redistribution of the nuclear m6A machinery is accompanied by a wide-scale reduction in the installation of m6A and other RNA modifications on both host and viral mRNAs. These results reveal a far-reaching mechanism by which HSV-1 subverts host gene expression to favor viral replication.
Assuntos
Herpesvirus Humano 1/fisiologia , Metiltransferases/metabolismo , RNA Mensageiro/metabolismo , Replicação Viral/fisiologia , Animais , Proteínas de Ciclo Celular , Linhagem Celular , Chlorocebus aethiops , Regulação Enzimológica da Expressão Gênica , Humanos , Metiltransferases/genética , Interferência de RNA , Processamento Pós-Transcricional do RNA , Fatores de Processamento de RNA , RNA Mensageiro/genética , RNA-Seq/métodos , Células VeroRESUMO
N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human ß-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m6A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m6A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m6A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m6A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m6A-modified and host m6A pathway components control ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.
Assuntos
Coronavirus Humano OC43/fisiologia , Processamento Pós-Transcricional do RNA/genética , SARS-CoV-2/fisiologia , Replicação Viral/genética , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Linhagem Celular , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Proteínas do Nucleocapsídeo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
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, here we profile 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.
Assuntos
Adenosina/análogos & derivados , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/genética , Splicing de RNA , RNA Viral/metabolismo , Células A549 , Adenosina/metabolismo , Adenovírus Humanos/patogenicidade , DNA Viral/genética , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Viral/genética , Análise de Sequência de RNA , Replicação ViralRESUMO
The genomes of DNA viruses encode deceptively complex transcriptomes evolved to maximize coding potential within the confines of a relatively small genome. Defining the full range of viral RNAs produced during an infection is key to understanding the viral replication cycle and its interactions with the host cell. Traditional short-read (Illumina) sequencing approaches are problematic in this setting due to the difficulty of assigning short reads to individual RNAs in regions of transcript overlap and to the biases introduced by the required recoding and amplification steps. Additionally, different methodologies may be required to analyze the 5' and 3' ends of RNAs, which increases both cost and effort. The advent of long-read nanopore sequencing simplifies this approach by providing a single assay that captures and sequences full length RNAs, either in cDNA or native RNA form. The latter is particularly appealing as it reduces known recoding biases whilst allowing more advanced analyses such as estimation of poly(A) tail length and the detection of RNA modifications including N6 -methyladenosine. Using herpes simplex virus (HSV)-infected primary fibroblasts as a template, we provide a step-by-step guide to the production of direct RNA sequencing libraries suitable for sequencing using Oxford Nanopore Technologies platforms and provide a simple computational approach to deriving a high-quality annotation of the HSV transcriptome from the resulting sequencing data. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Productive infection of primary fibroblasts with herpes simplex virus Support Protocol: Cell passage and plating of primary fibroblasts Basic Protocol 2: Preparation and sequencing of dRNA-seq libraries from virus-infected cells Basic Protocol 3: Processing, alignment, and analysis of dRNA-seq datasets.
Assuntos
Técnicas de Cultura de Células/métodos , Herpes Simples/virologia , Sequenciamento por Nanoporos/métodos , Análise de Sequência de RNA/métodos , Simplexvirus/genética , Cultura de Vírus/métodos , Fibroblastos/virologia , Humanos , RNA Viral/genética , Simplexvirus/fisiologia , TranscriptomaRESUMO
Meeting Report on the 9th Annual Symposium of the Colorado Alphaherpesvirus Latency Society (CALS) held on May 8-11, 2019, in Vail, CO.
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Alphaherpesvirinae/fisiologia , Infecções por Herpesviridae/virologia , Latência Viral , Colorado , Humanos , Sociedades MédicasRESUMO
We describe a primary neuronal culture system suitable for molecular characterization of herpes simplex virus type 1 (HSV-1) infection, latency, and reactivation. While several alternative models are available, including infections of live animal or explanted ganglia, these are complicated by the presence of multiple cell types, including immune cells, and difficulties in manipulating the neuronal environment. The highly pure neuron culture system described here can be readily manipulated and is ideal for molecular studies that focus exclusively on the relationship between the virus and host neuron, the fundamental unit of latency. As such this model allows for detailed investigations of both viral and neuronal factors involved in the establishment and maintenance of HSV-1 latency and in viral reactivation induced by defined stimuli.
Assuntos
Técnicas de Cultura de Células , Herpesvirus Humano 1/fisiologia , Neurônios , Ativação Viral/fisiologia , Latência Viral/fisiologia , Animais , Células Cultivadas , Neurônios/metabolismo , Neurônios/patologia , Neurônios/virologia , Ratos , Ratos Sprague-DawleyRESUMO
The mTOR pathway integrates both extracellular and intracellular signals and serves as a central regulator of cell metabolism, growth, survival, and stress responses. Neurotropic viruses, such as herpes simplex virus-1 (HSV-1), also rely on cellular AKT-mTORC1 signaling to achieve viral latency. Here, we define a novel genotoxic response whereby spatially separated signals initiated by extracellular neurotrophic factors and nuclear DNA damage are integrated by the AKT-mTORC1 pathway. We demonstrate that endogenous DNA double-strand breaks (DSBs) mediated by Topoisomerase 2ß-DNA cleavage complex (TOP2ßcc) intermediates are required to achieve AKT-mTORC1 signaling and maintain HSV-1 latency in neurons. Suppression of host DNA-repair pathways that remove TOP2ßcc trigger HSV-1 reactivation. Moreover, perturbation of AKT phosphorylation dynamics by downregulating the PHLPP1 phosphatase led to AKT mis-localization and disruption of DSB-induced HSV-1 reactivation. Thus, the cellular genome integrity and environmental inputs are consolidated and co-opted by a latent virus to balance lifelong infection with transmission.
Assuntos
DNA Topoisomerases Tipo II/genética , Herpesvirus Humano 1/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-akt/genética , Latência Viral/genética , Animais , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Herpesvirus Humano 1/patogenicidade , Humanos , Proteína Homóloga a MRE11/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Neurônios/metabolismo , Neurônios/virologia , Fosforilação , Ratos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genéticaRESUMO
Characterizing complex viral transcriptomes by conventional RNA sequencing approaches is complicated by high gene density, overlapping reading frames, and complex splicing patterns. Direct RNA sequencing (direct RNA-seq) using nanopore arrays offers an exciting alternative whereby individual polyadenylated RNAs are sequenced directly, without the recoding and amplification biases inherent to other sequencing methodologies. Here we use direct RNA-seq to profile the herpes simplex virus type 1 (HSV-1) transcriptome during productive infection of primary cells. We show how direct RNA-seq data can be used to define transcription initiation and RNA cleavage sites associated with all polyadenylated viral RNAs and demonstrate that low level read-through transcription produces a novel class of chimeric HSV-1 transcripts, including a functional mRNA encoding a fusion of the viral E3 ubiquitin ligase ICP0 and viral membrane glycoprotein L. Thus, direct RNA-seq offers a powerful method to characterize the changing transcriptional landscape of viruses with complex genomes.