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IMPORTANCE: The current view is that the default pathway of Kaposi's sarcoma-associated herpesvirus (KSHV) infection is the establishment of latency, which is a prerequisite for lifelong infection and viral oncogenesis. This view about KSHV infection is supported by the observations that KSHV latently infects most of the cell lines cultured in vitro in the absence of any environmental stresses that may occur in vivo. The goal of this study was to determine the effect of hypoxia, a natural stress stimulus, on primary KSHV infection. Our data indicate that hypoxia promotes euchromatin formation on the KSHV genome following infection and supports lytic de novo KSHV infection. We also discovered that hypoxia-inducible factor-1α is required and sufficient for allowing lytic KSHV infection. Based on our results, we propose that hypoxia promotes lytic de novo infection in cells that otherwise support latent infection under normoxia; that is, the environmental conditions can determine the outcome of KSHV primary infection.
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Infecciones por Herpesviridae , Subunidad alfa del Factor 1 Inducible por Hipoxia , Hipoxia , Humanos , Regulación Viral de la Expresión Génica , Herpesvirus Humano 8 , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Sarcoma de Kaposi , Latencia del VirusRESUMEN
BACKGROUND: Epstein-Barr virus (EBV) is an important human pathogenic gammaherpesvirus with carcinogenic potential. The EBV transcriptome has previously been analyzed using both Illumina-based short read-sequencing and Pacific Biosciences RS II-based long-read sequencing technologies. Since the various sequencing methods have distinct strengths and limitations, the use of multiplatform approaches have proven to be valuable. The aim of this study is to provide a more complete picture on the transcriptomic architecture of EBV. METHODS: In this work, we apply the Oxford Nanopore Technologies MinION (long-read sequencing) platform for the generation of novel transcriptomic data, and integrate these with other's data generated by another LRS approach, Pacific BioSciences RSII sequencing and Illumina CAGE-Seq and Poly(A)-Seq approaches. Both amplified and non-amplified cDNA sequencings were applied for the generation of sequencing reads, including both oligo-d(T) and random oligonucleotide-primed reverse transcription. EBV transcripts are identified and annotated using the LoRTIA software suite developed in our laboratory. RESULTS: This study detected novel genes embedded into longer host genes containing 5'-truncated in-frame open reading frames, which potentially encode N-terminally truncated proteins. We also detected a number of novel non-coding RNAs and transcript length isoforms encoded by the same genes but differing in their start and/or end sites. This study also reports the discovery of novel splice isoforms, many of which may represent altered coding potential, and of novel replication-origin-associated transcripts. Additionally, novel mono- and multigenic transcripts were identified. An intricate meshwork of transcriptional overlaps was revealed. CONCLUSIONS: An integrative approach applying multi-technique sequencing technologies is suitable for reliable identification of complex transcriptomes because each techniques has different advantages and limitations, and the they can be used for the validation of the results obtained by a particular approach.
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Infecciones por Virus de Epstein-Barr , Transcriptoma , Infecciones por Virus de Epstein-Barr/genética , Perfilación de la Expresión Génica , Herpesvirus Humano 4/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Sistemas de Lectura AbiertaRESUMEN
BACKGROUND: Alternative polyadenylation is commonly examined using cDNA sequencing, which is known to be affected by template-switching artifacts. However, the effects of such template-switching artifacts on alternative polyadenylation are generally disregarded, while alternative polyadenylation artifacts are attributed to internal priming. RESULTS: Here, we analyzed both long-read cDNA sequencing and direct RNA sequencing data of two organisms, generated by different sequencing platforms. We developed a filtering algorithm which takes into consideration that template-switching can be a source of artifactual polyadenylation when filtering out spurious polyadenylation sites. The algorithm outperformed the conventional internal priming filters based on comparison to direct RNA sequencing data. We also showed that the polyadenylation artifacts arise in cDNA sequencing at consecutive stretches of as few as three adenines. There was no substantial difference between the lengths of poly(A) tails at the artifactual and the true transcriptional end sites even though it is expected that internal priming artifacts have shorter poly(A) tails than genuine polyadenylated reads. CONCLUSIONS: Our findings suggest that template switching plays an important role in the generation of spurious polyadenylation and support the need for more rigorous filtering of artifactual polyadenylation sites in cDNA data, or that alternative polyadenylation should be annotated using native RNA sequencing.
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Poliadenilación , Artefactos , ADN Complementario/genética , Análisis de Secuencia de ADN , Transcripción GenéticaRESUMEN
The role of RNA molecules in the priming of DNA replication and in providing a template for telomerase extension has been known for decades. Since then, several transcripts have been discovered, which play diverse roles in governing replication, including regulation of RNA primer formation, the recruitment of replication origin (Ori) recognition complex, and the assembly of replication fork. Recent studies on viral transcriptomes have revealed novel classes of replication-associated (ra)RNAs, which are expressed from the genomic locations in close vicinity to the Ori. Many of them overlap the Ori, whereas others are terminated close to the replication origin. These novel transcripts can be both protein-coding and non-coding RNAs. The Ori-overlapping part of the mRNAs is generally either the 5'-untranslated regions (UTRs), or the 3'-UTRs of the longer isoforms. Several raRNAs have been identified in various viral families using primarily third-generation long-read sequencing. Hyper-editing of these transcripts has also been described.
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Regulación Viral de la Expresión Génica , Transcripción Genética , Fenómenos Fisiológicos de los Virus , Replicación Viral/genética , Virus/genética , Animales , Epistasis Genética , Eucariontes , Redes Reguladoras de Genes , Humanos , Células Procariotas , Unión Proteica , Interferencia de ARNRESUMEN
The temporal coordination of viral gene expression is imperative for the regulation of the herpesvirus replication cycle. While the main factors of this transcriptional coordination are known, the subtler control mechanisms of gene expression remain elusive. Recent long read sequencing-based approached have revealed an intricate meshwork of overlaps between the herpesvirus transcripts and the overlap of the replication origins with noncoding RNAs. It has been shown that the transcriptional apparatuses can physically interfere with one another while transcribing overlapping regions. We hypothesize that transcriptional interference regulates the global gene expression across the herpesvirus genome. Additionally, an overall decrease in transcriptional activity in individual viral genes has been observed following the onset of DNA replication. An overlap of the replication origins with specific transcripts has also been described in several herpesviruses. The genome-wide interactions between the transcriptional apparatuses and between the replication and transcriptional machineries suggest the existence of novel layers of genetic regulation.
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ADN Viral/biosíntesis , Herpesviridae/genética , ARN Viral/biosíntesis , Origen de Réplica/genética , Replicación del ADN/genética , ADN Viral/genética , Regulación Viral de la Expresión Génica , Redes Reguladoras de Genes/genética , Genoma Viral/genética , ARN Viral/genéticaRESUMEN
Congenital muscular dystrophy (CMD), a subgroup of myopathies is a genetically and clinically heterogeneous group of inherited muscle disorders and is characterized by progressive muscle weakness, fiber size variability, fibrosis, clustered necrotic fibers, and central myonuclei present in regenerating muscle. Type IV collagen (COL4A1) mutations have recently been identified in patients with intracerebral, vascular, renal, ophthalmologic pathologies and congenital muscular dystrophy, consistent with diagnoses of Walker-Warburg Syndrome or Muscle-Eye-Brain disease. Morphological characteristics of muscular dystrophy have also been demonstrated Col4a1 mutant mice. Yet, several aspects of the pathomechanism of COL4A1-associated muscle defects remained largely uncharacterized. Based on the results of genetic, histological, molecular, and biochemical analyses in an allelic series of Drosophila col4a1 mutants, we provide evidence that col4a1 mutations arise by transitions in glycine triplets, associate with severely compromised muscle fibers within the single-layer striated muscle of the common oviduct, characterized by loss of sarcomere structure, disintegration and streaming of Z-discs, indicating an essential role for the COL4A1 protein. Features of altered cytoskeletal phenotype include actin bundles traversing over sarcomere units, amorphous actin aggregates, atrophy, and aberrant fiber size. The mutant COL4A1-associated defects appear to recapitulate integrin-mediated adhesion phenotypes observed in RNA-inhibitory Drosophila. Our results provide insight into the mechanistic details of COL4A1-associated muscle disorders and suggest a role for integrin-collagen interaction in the maintenance of sarcomeres.
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Colágeno Tipo IV/metabolismo , Drosophila/metabolismo , Integrinas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Animales , Biomarcadores , Adhesión Celular/genética , Cromosomas , Colágeno Tipo IV/genética , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Técnica del Anticuerpo Fluorescente , Mutación , Sarcómeros/metabolismo , Sarcómeros/ultraestructuraRESUMEN
BACKGROUND: Pseudorabies virus (PRV) is the causative agent of Aujeszky's disease giving rise to significant economic losses worldwide. Many countries have implemented national programs for the eradication of this virus. In this study, long-read sequencing was used to determine the nucleotide sequence of the genome of a novel PRV strain (PRV-MdBio) isolated in Serbia. RESULTS: In this study, a novel PRV strain was isolated and characterized. PRV-MdBio was found to exhibit similar growth properties to those of another wild-type PRV, the strain Kaplan. Single-molecule real-time (SMRT) sequencing has revealed that the new strain differs significantly in base composition even from strain Kaplan, to which it otherwise exhibits the highest similarity. We compared the genetic composition of PRV-MdBio to strain Kaplan and the China reference strain Ea and obtained that radical base replacements were the most common point mutations preceding conservative and silent mutations. We also found that the adaptation of PRV to cell culture does not lead to any tendentious genetic alteration in the viral genome. CONCLUSION: PRV-MdBio is a wild-type virus, which differs in base composition from other PRV strains to a relatively large extent.
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BACKGROUND: Understanding the underlying genetic structure of human populations is of fundamental interest to both biological and social sciences. Advances in high-throughput genotyping technology have markedly improved our understanding of global patterns of human genetic variation. The most widely used methods for collecting variant information at the DNA-level include whole genome sequencing, which remains costly, and the more economical solution of array-based techniques, as these are capable of simultaneously genotyping a pre-selected set of variable DNA sites in the human genome. The largest publicly accessible set of human genomic sequence data available today originates from exome sequencing that comprises around 1.2% of the whole genome (approximately 30 million base pairs). RESULTS: To unbiasedly compare the effect of SNP selection strategies in population genetic analysis we subsampled the variants of the same highly curated 1 K Genome dataset to mimic genome, exome sequencing and array data in order to eliminate the effect of different chemistry and error profiles of these different approaches. Next we compared the application of the exome dataset to the array-based dataset and to the gold standard whole genome dataset using the same population genetic analysis methods. CONCLUSIONS: Our results draw attention to some of the inherent problems that arise from using pre-selected SNP sets for population genetic analysis. Additionally, we demonstrate that exome sequencing provides a better alternative to the array-based methods for population genetic analysis. In this study, we propose a strategy for unbiased variant collection from exome data and offer a bioinformatics protocol for proper data processing.
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Genética de Población , Genoma Humano , Genómica , Bases de Datos Genéticas , Evolución Molecular , Marcadores Genéticos , Variación Genética , Genómica/métodos , Humanos , Polimorfismo de Nucleótido Simple , Secuenciación del ExomaRESUMEN
BACKGROUND: Varicella zoster virus (VZV) is a human pathogenic alphaherpesvirus harboring a relatively large DNA molecule. The VZV transcriptome has already been analyzed by microarray and short-read sequencing analyses. However, both approaches have substantial limitations when used for structural characterization of transcript isoforms, even if supplemented with primer extension or other techniques. Among others, they are inefficient in distinguishing between embedded RNA molecules, transcript isoforms, including splice and length variants, as well as between alternative polycistronic transcripts. It has been demonstrated in several studies that long-read sequencing is able to circumvent these problems. RESULTS: In this work, we report the analysis of the VZV lytic transcriptome using the Oxford Nanopore Technologies sequencing platform. These investigations have led to the identification of 114 novel transcripts, including mRNAs, non-coding RNAs, polycistronic RNAs and complex transcripts, as well as 10 novel spliced transcripts and 25 novel transcription start site isoforms and transcription end site isoforms. A novel class of transcripts, the nroRNAs are described in this study. These transcripts are encoded by the genomic region located in close vicinity to the viral replication origin. We also show that the ORF63 exhibits a complex structural variation encompassing the splice sites of VZV latency transcripts. Additionally, we have detected RNA editing in a novel non-coding RNA molecule. CONCLUSIONS: Our investigations disclosed a composite transcriptomic architecture of VZV, including the discovery of novel RNA molecules and transcript isoforms, as well as a complex meshwork of transcriptional read-throughs and overlaps. The results represent a substantial advance in the annotation of the VZV transcriptome and in understanding the molecular biology of the herpesviruses in general.
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Herpesvirus Humano 3/genética , Transcriptoma , Línea Celular , Humanos , Sistemas de Lectura Abierta/genética , Isoformas de Proteínas/genética , Edición de ARN , Empalme del ARN , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN Viral/aislamiento & purificación , ARN Viral/metabolismo , Análisis de Secuencia de ADN , Sitio de Iniciación de la Transcripción , Proteínas Virales/genéticaRESUMEN
Neurotropic herpesviruses can establish lifelong infection in humans and contribute to severe diseases including encephalitis and neurodegeneration. However, the mechanisms through which the brain's immune system recognizes and controls viral infections propagating across synaptically linked neuronal circuits have remained unclear. Using a well-established model of alphaherpesvirus infection that reaches the brain exclusively via retrograde transsynaptic spread from the periphery, and in vivo two-photon imaging combined with high resolution microscopy, we show that microglia are recruited to and isolate infected neurons within hours. Selective elimination of microglia results in a marked increase in the spread of infection and egress of viral particles into the brain parenchyma, which are associated with diverse neurological symptoms. Microglia recruitment and clearance of infected cells require cell-autonomous P2Y12 signalling in microglia, triggered by nucleotides released from affected neurons. In turn, we identify microglia as key contributors to monocyte recruitment into the inflamed brain, which process is largely independent of P2Y12. P2Y12-positive microglia are also recruited to infected neurons in the human brain during viral encephalitis and both microglial responses and leukocyte numbers correlate with the severity of infection. Thus, our data identify a key role for microglial P2Y12 in defence against neurotropic viruses, whilst P2Y12-independent actions of microglia may contribute to neuroinflammation by facilitating monocyte recruitment to the sites of infection.
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Encéfalo/metabolismo , Infecciones por Herpesviridae/metabolismo , Microglía/metabolismo , Monocitos/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Transducción de Señal/fisiología , Animales , Encéfalo/virología , Ratones , Microglía/virología , Neuronas/metabolismo , Neuronas/virologíaRESUMEN
Pseudorabies virus (PRV) is an animal alphaherpesvirus with a wide host range. PRV has 67 protein-coding genes and several non-coding RNA molecules, which can be classified into three temporal groups, immediate early, early and late classes. The ul54 gene of PRV and its homolog icp27 of herpes simplex virus have a multitude of functions, including the regulation of viral DNA synthesis and the control of the gene expression. Therefore, abrogation of PRV ul54 function was expected to exert a significant effect on the global transcriptome and on DNA replication. Real-time PCR and real-time RT-PCR platforms were used to investigate these presumed effects. Our analyses revealed a drastic impact of the ul54 mutation on the genome-wide expression of PRV genes, especially on the transcription of the true late genes. A more than two hour delay was observed in the onset of DNA replication, and the amount of synthesized DNA molecules was significantly decreased in comparison to the wild-type virus. Furthermore, in this work, we were able to successfully demonstrate the utility of long-read SMRT sequencing for genotyping of mutant viruses.
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Replicación del ADN/fisiología , ADN Viral/fisiología , Eliminación de Gen , Herpesvirus Suido 1/fisiología , Animales , Línea Celular , Genotipo , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Proteínas ViralesRESUMEN
The pseudorabies virus (PRV; also known as Suid herpesvirus-1) is a neurotropic herpesvirus of swine. The us7 and us8 genes of this virus encode the glycoprotein I and E membrane proteins that form a heterodimer that is known to control cell-to-cell spread in tissue culture and in animals. In this study, we investigated the effect of the deletion of the PRV us7 and us8 genes on the genome-wide transcription and DNA replication using a multi-time-point quantitative reverse transcriptase-based real-time PCR technique. Abrogation of the us7/8 gene function was found to exert a drastic but differential effect on the expression of PRV genes during lytic infection. In the mutant virus, all kinetic classes of viral genes were significantly down-regulated at the first 6 h of infection, while having been upregulated later. The level of upregulation was the highest in the immediate-early (IE) and the early (E) genes; lower in the early-late (E/L) genes; and the lowest in the late (L) genes. The relative contribution of the L transcripts to the global transcriptome became lower, while the rest of the transcripts were expressed at a higher level in the mutant than in the wild-type virus.
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Eliminación de Gen , Regulación Viral de la Expresión Génica , Herpesvirus Suido 1/genética , Seudorrabia/virología , Enfermedades de los Porcinos/virología , Proteínas Virales/genética , Animales , Replicación del ADN , Herpesvirus Suido 1/metabolismo , Porcinos , Proteínas Virales/metabolismoRESUMEN
BACKGROUND: Pseudorabies virus is a widely-studied model organism of the Herpesviridae family, with a compact genome arrangement of 72 known coding sequences. In order to obtain an up-to-date genetic map of the virus, a combination of RNA-sequencing approaches were applied, as recent advancements in high-throughput sequencing methods have provided a wealth of information on novel RNA species and transcript isoforms, revealing additional layers of transcriptome complexity in several viral species. RESULTS: The total RNA content and polyadenylation landscape of pseudorabies virus were characterized for the first time at high coverage by Illumina high-throughput sequencing of cDNA samples collected during the lytic infectious cycle. As anticipated, nearly all of the viral genome was transcribed, with the exception of loci in the large internal and terminal repeats, and several small intergenic repetitive sequences. Our findings included a small novel polyadenylated non-coding RNA near an origin of replication, and the single-base resolution mapping of 3' UTRs across the viral genome. Alternative polyadenylation sites were found in a number of genes and a novel alternative splice site was characterized in the ep0 gene, while previously known splicing events were confirmed, yielding no alternative splice isoforms. Additionally, we detected the active polyadenylation of transcripts earlier believed to be transcribed as part of polycistronic RNAs. CONCLUSION: To the best of our knowledge, the present work has furnished the highest-resolution transcriptome map of an alphaherpesvirus to date, and reveals further complexities of viral gene expression, with the identification of novel transcript boundaries, alternative splicing of the key transactivator EP0, and a highly abundant, novel non-coding RNA near the lytic replication origin. These advances provide a detailed genetic map of PRV for future research.
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Perfilación de la Expresión Génica/métodos , Herpesvirus Suido 1/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ARN/métodos , Genoma Viral , Datos de Secuencia Molecular , Poliadenilación , Sitios de Empalme de ARN , ARN Mensajero/análisis , ARN Viral/análisisRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) is a large, oncogenic DNA virus belonging to the gammaherpesvirus subfamily. KSHV has been extensively studied with various high-throughput RNA-sequencing approaches to map the transcription start and end sites, the splice junctions, and the translation initiation sites. Despite these efforts, the comprehensive annotation of the viral transcriptome remains incomplete. In the present study, we generated a long-read sequencing data set of the lytic and latent KSHV transcriptome using native RNA and direct cDNA-sequencing methods. This was supplemented with Cap Analysis of Gene Expression sequencing based on a short-read platform. We also utilized data sets from previous publications for our analysis. As a result of this combined approach, we have identified a number of novel viral transcripts and RNA isoforms and have either corroborated or improved the annotation of previously identified viral RNA molecules, thereby notably enhancing our comprehension of the transcriptomic architecture of the KSHV genome. We also evaluated the coding capability of transcripts previously thought to be non-coding by integrating our data on the viral transcripts with translatomic information from other publications.IMPORTANCEDeciphering the viral transcriptome of Kaposi's sarcoma-associated herpesvirus is of great importance because we can gain insight into the molecular mechanism of viral replication and pathogenesis, which can help develop potential targets for antiviral interventions. Specifically, the identification of substantial transcriptional overlaps by this work suggests the existence of a genome-wide interference between transcriptional machineries. This finding indicates the presence of a novel regulatory layer, potentially controlling the expression of viral genes.
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Herpesvirus Humano 8 , Herpesvirus Humano 8/genética , Transcriptoma/genética , Replicación Viral/genética , Perfilación de la Expresión Génica , ARN/metabolismoRESUMEN
In this study, we employed short- and long-read sequencing technologies to delineate the transcriptional architecture of the human monkeypox virus and to identify key regulatory elements that govern its gene expression. Specifically, we conducted a transcriptomic analysis to annotate the transcription start sites (TSSs) and transcription end sites (TESs) of the virus by utilizing Cap Analysis of gene expression sequencing on the Illumina platform and direct RNA sequencing on the Oxford Nanopore technology device. Our investigations uncovered significant complexity in the use of alternative TSSs and TESs in viral genes. In this research, we also detected the promoter elements and poly(A) signals associated with the viral genes. Additionally, we identified novel genes in both the left and right variable regions of the viral genome.IMPORTANCEGenerally, gaining insight into how the transcription of a virus is regulated offers insights into the key mechanisms that control its life cycle. The recent outbreak of the human monkeypox virus has underscored the necessity of understanding the basic biology of its causative agent. Our results are pivotal for constructing a comprehensive transcriptomic atlas of the human monkeypox virus, providing valuable resources for future studies.
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BACKGROUND: Pseudorabies virus (PRV), an alpha-herpesvirus of swine, is a widely used model organism in investigations of the molecular pathomechanisms of the herpesviruses. This work is the continuation of our earlier studies, in which we investigated the effect of the abrogation of gene function on the viral transcriptome by knocking out PRV genes playing roles in the coordination of global gene expression of the virus. In this study, we deleted the us1 gene encoding the ICP22, an important viral regulatory protein, and analyzed the changes in the expression of other PRV genes. RESULTS: A multi-timepoint real-time RT-PCR technique was applied to evaluate the impact of deletion of the PRV us1 gene on the overall transcription kinetics of viral genes. The mutation proved to exert a differential effect on the distinct kinetic classes of PRV genes at the various stages of lytic infection. In the us1 gene-deleted virus, all the kinetic classes of the genes were significantly down-regulated in the first hour of infection. After 2 to 6 h of infection, the late genes were severely suppressed, whereas the early genes were unaffected. In the late stage of infection, the early genes were selectively up-regulated. In the mutant virus, the transcription of the ie180 gene, the major coordinator of PRV gene expression, correlated closely with the transcription of other viral genes, a situation which was not found in the wild-type (wt) virus. A 4-h delay was observed in the commencement of DNA replication in the mutant virus as compared with the wt virus. The rate of transcription from a gene normalized to the relative copy number of the viral genome was observed to decline drastically following the initiation of DNA replication in both the wt and mutant backgrounds. Finally, the switch between the expressions of the early and late genes was demonstrated not to be controlled by DNA replication, as is widely believed, since the switch preceded the DNA replication. CONCLUSIONS: Our results show a strong dependence of PRV gene expression on the presence of functional us1 gene. ICP22 is shown to exert a differential effect on the distinct kinetic classes of PRV genes and to disrupt the close correlation between the transcription kinetics of ie180 and other PRV transcripts. Furthermore, DNA replication exerts a severe constraint on the viral transcription.
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Regulación Viral de la Expresión Génica , Herpesvirus Suido 1/genética , Proteínas Inmediatas-Precoces/metabolismo , Transcripción Genética , Herpesvirus Suido 1/química , Herpesvirus Suido 1/metabolismo , Proteínas Inmediatas-Precoces/genética , Cinética , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) is a large, oncogenic DNA virus belonging to the gammaherpesvirus subfamily. KSHV has been extensively studied with various high-throughput RNA-sequencing approaches to map the transcription start and end sites, the splice junctions, and the translation initiation sites. Despite these efforts, the comprehensive annotation of the viral transcriptome remains incomplete. In the present study, we generated a long-read sequencing dataset of the lytic and latent KSHV transcriptome using native RNA and direct cDNA sequencing methods. This was supplemented with CAGE sequencing based on a short-read platform. We also utilized datasets from previous publications for our analysis. As a result of this combined approach, we have identified a number of novel viral transcripts and RNA isoforms and have either corroborated or improved the annotation of previously identified viral RNA molecules, thereby notably enhancing our comprehension of the transcriptomic architecture of the KSHV genome. We also evaluated the coding capability of transcripts previously thought to be non-coding, by integrating our data on the viral transcripts with translatomic information from other publications.
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This study employed both short-read sequencing (SRS, Illumina) and long-read sequencing (LRS Oxford Nanopore Technologies) platforms to conduct a comprehensive analysis of the equid alphaherpesvirus 1 (EHV-1) transcriptome. The study involved the annotation of canonical mRNAs and their transcript variants, encompassing transcription start site (TSS) and transcription end site (TES) isoforms, in addition to alternative splicing forms. Furthermore, the study revealed the presence of numerous non-coding RNA (ncRNA) molecules, including intergenic and antisense transcripts, produced by EHV-1. An intriguing finding was the abundant production of chimeric transcripts, some of which potentially encode fusion polypeptides. Moreover, EHV-1 exhibited a greater incidence of transcriptional overlaps and splicing compared to related viruses. It is noteworthy that many genes have their unique TESs along with the co-terminal transcription ends, a characteristic scarcely seen in other alphaherpesviruses. The study also identified transcripts that overlap the replication origins of the virus. Moreover, a novel ncRNA, referred to as NOIR, was found to intersect with the 5'-ends of longer transcript isoform specified by the major transactivator genes ORF64 and ORF65, surrounding the OriL. These findings together imply the existence of a key regulatory mechanism that governs both transcription and replication through, among others, a process that involves interference between the DNA and RNA synthesis machineries.
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Long-read sequencing (LRS) techniques enable the identification of full-length RNA molecules in a single run eliminating the need for additional assembly steps. LRS research has exposed unanticipated transcriptomic complexity in various organisms, including viruses. Herpesviruses are known to produce a range of transcripts, either close to or overlapping replication origins (Oris) and neighboring genes related to transcription or replication, which possess confirmed or potential regulatory roles. In our research, we employed both new and previously published LRS and short-read sequencing datasets to uncover additional Ori-proximal transcripts in nine herpesviruses from all three subfamilies (alpha, beta and gamma). We discovered novel long non-coding RNAs, as well as splice and length isoforms of mRNAs. Moreover, our analysis uncovered an intricate network of transcriptional overlaps within the examined genomic regions. We demonstrated that herpesviruses display distinct patterns of transcriptional overlaps in the vicinity of or at the Oris. Our findings suggest the existence of a 'super regulatory center' in the genome of alphaherpesviruses that governs the initiation of both DNA replication and global transcription through multilayered interactions among the molecular machineries.
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Herpesviridae , Origen de Réplica , Origen de Réplica/genética , Herpesviridae/genética , Transcriptoma , Perfilación de la Expresión Génica , GenómicaRESUMEN
The recent human Monkeypox outbreak underlined the importance of studying basic biology of orthopoxviruses. However, the transcriptome of its causative agent has not been investigated before neither with short-, nor with long-read sequencing approaches. This Oxford Nanopore long-read RNA-Sequencing dataset fills this gap. It will enable the in-depth characterization of the transcriptomic architecture of the monkeypox virus, and may even make possible to annotate novel host transcripts. Moreover, our direct cDNA and native RNA sequencing reads will allow the estimation of gene expression changes of both the virus and the host cells during the infection. Overall, our study will lead to a deeper understanding of the alterations caused by the viral infection on a transcriptome level.