Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 49
Filtrar
Mais filtros










Intervalo de ano de publicação
1.
Artigo em Inglês | MEDLINE | ID: mdl-34374828

RESUMO

This chapter first details the structure, organization and coding content of the VZV genome to provide a foundation on which the molecular evolution of the virus can be projected. We subsequently describe the evolution of molecular profiling approaches from restriction fragment length polymorphisms to single nucleotide polymorphism profiling to modern day high-throughput sequencing approaches. We describe how the application of these methodologies led to our current model of VZV phylogeograpy including the number and structure of geographic clades and the role of recombination in reshaping these.

2.
Proc Natl Acad Sci U S A ; 118(30)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34282019

RESUMO

N 6-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.

3.
Genes Dev ; 35(13-14): 1005-1019, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34168039

RESUMO

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 fármacos
4.
Cell Host Microbe ; 29(6): 1014-1029.e8, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-33894129

RESUMO

The contributions of the viral component of the microbiome-the virome-to the development of innate and adaptive immunity are largely unknown. Here, we systematically defined the host response in mice to a panel of eukaryotic enteric viruses representing six different families. Infections with most of these viruses were asymptomatic in the mice, the magnitude and duration of which was dependent on the microbiota. Flow cytometric and transcriptional profiling of mice mono-associated with these viruses unveiled general adaptations by the host, such as lymphocyte differentiation and IL-22 signatures in the intestine, as well as numerous viral-strain-specific responses that persisted. Comparison with a dataset derived from analogous bacterial mono-association in mice identified bacterial species that evoke an immune response comparable with the viruses we examined. These results expand an understanding of the immune space occupied by the enteric virome and underscore the importance of viral exposure events.

5.
Sci Rep ; 11(1): 4586, 2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33633271

RESUMO

Epstein-Barr virus (EBV) is one of the most common viruses latently infecting humans. Little is known about the impact of human genetic variation on the large inter-individual differences observed in response to EBV infection. To search for a potential imprint of host genomic variation on the EBV sequence, we jointly analyzed paired viral and human genomic data from 268 HIV-coinfected individuals with CD4 + T cell count < 200/mm3 and elevated EBV viremia. We hypothesized that the reactivated virus circulating in these patients could carry sequence variants acquired during primary EBV infection, thereby providing a snapshot of early adaptation to the pressure exerted on EBV by the individual immune response. We searched for associations between host and pathogen genetic variants, taking into account human and EBV population structure. Our analyses revealed significant associations between human and EBV sequence variation. Three polymorphic regions in the human genome were found to be associated with EBV variation: one at the amino acid level (BRLF1:p.Lys316Glu); and two at the gene level (burden testing of rare variants in BALF5 and BBRF1). Our findings confirm that jointly analyzing host and pathogen genomes can identify sites of genomic interactions, which could help dissect pathogenic mechanisms and suggest new therapeutic avenues.

6.
Nat Commun ; 11(1): 6324, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33303747

RESUMO

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. VZV transcription during latency is restricted to the latency-associated transcript (VLT) and RNA 63 (encoding ORF63) in naturally VZV-infected human trigeminal ganglia (TG). While significantly more abundant, VLT levels positively correlated with RNA 63 suggesting co-regulated transcription during latency. Here, we identify VLT-ORF63 fusion transcripts and confirm VLT-ORF63, but not RNA 63, expression in human TG neurons. During in vitro latency, VLT is transcribed, whereas VLT-ORF63 expression is induced by reactivation stimuli. One isoform of VLT-ORF63, encoding a fusion protein combining VLT and ORF63 proteins, induces broad viral gene transcription. 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.


Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 3/genética , Células Receptoras Sensoriais/virologia , Proteínas Virais/genética , Ativação Viral/genética , Latência Viral/genética , Anisomicina/farmacologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fases de Leitura Aberta/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética/efeitos dos fármacos , Gânglio Trigeminal/patologia , Gânglio Trigeminal/virologia , Proteínas Virais/metabolismo
7.
Nat Commun ; 11(1): 6016, 2020 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-33243990

RESUMO

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 Viral
8.
mBio ; 11(5)2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024035

RESUMO

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, noncanonical ORFs, and ORF fusions, as well as putative noncoding RNAs (ncRNAs). Furthermore, we determined the kinetic class of all VZV transcripts and observed, unexpectedly, that transcripts encoding the ORF62 protein, previously designated 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.IMPORTANCE Transcription from herpesviral genomes, executed by the host RNA polymerase II and regulated by viral proteins, results in coordinated viral gene expression to efficiently produce infectious progeny. However, the complete coding potential and regulation of viral gene expression remain ill-defined for the human alphaherpesvirus varicella-zoster virus (VZV), causative agent of both varicella and herpes zoster. Here, we present a comprehensive overview of the VZV transcriptome and the kinetic class of all identified viral transcripts, using two virus strains and two biologically relevant cell types. Additionally, our data provide an overview of how VZV diversifies its transcription from one of the smallest herpesviral genomes. Unexpectedly, the transcript encoding the major viral transactivator protein (pORF62) was expressed with Late kinetics, whereas orthologous transcripts in other alphaherpesviruses are typically expressed during the immediate early phase. Therefore, our work both establishes the architecture of the VZV transcriptome and provides insight into regulation of alphaherpesvirus gene expression.

9.
Virology ; 546: 38-50, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32452416

RESUMO

The varicella-zoster virus (VZV) genome, comprises both unique and repeated regions. The genome also includes reiteration regions, designated R1 to R5, which are tandemly repeating sequences termed elements. These regions represent an understudied feature of the VZV genome. The R4 region is duplicated, with one copy in the internal repeat short (IRs) which we designated R4A and a second copy in the terminal repeat short (TRs) termed R4B. We developed primers to amplify and Sanger sequence these regions, including independent amplification of both R4 regions. Reiteration regions from >80 cases of PCR-confirmed shingles were sequenced and analyzed. Complete genome sequences for the remaining portions of these viruses were determined using Illumina MiSeq. We identified 28 elements not previously reported, including at least one element for each R region. Length heterogeneity was substantial in R3, R4A and R4B. Length heterogeneity between the two copies of R4 was common.


Assuntos
Genoma Viral , Herpes Zoster/virologia , Herpesvirus Humano 3/genética , Sequências de Repetição em Tandem , DNA Viral/genética , Herpesvirus Humano 3/metabolismo , Humanos , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas Virais/genética , Proteínas Virais/metabolismo
10.
Curr Protoc Microbiol ; 57(1): e99, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32255550

RESUMO

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 , Transcriptoma
11.
Virus Evol ; 6(1): veaa012, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32099667

RESUMO

Herpes Simplex Virus type 1 (HSV-1) chronically infects over 70 per cent 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. In 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.

12.
J Invest Dermatol ; 140(4): 774-784.e11, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31626786

RESUMO

Varicella zoster virus (VZV) is a skin-tropic virus that infects epidermal keratinocytes and causes chickenpox. Although common, VZV infection can be life-threatening, particularly in the immunocompromized. Therefore, understanding VZV-keratinocyte interactions is important to find new treatments beyond vaccination and antiviral drugs. In VZV-infected skin, kallikrein 6 and the ubiquitin ligase MDM2 are upregulated concomitant with keratin 10 (KRT10) downregulation. MDM2 binds to KRT10, targeting it for degradation via the ubiquitin-proteasome pathway. Preventing KRT10 degradation reduced VZV propagation in culture and prevented epidermal disruption in skin explants. KRT10 knockdown induced expression of NR4A1 and enhanced viral propagation in culture. NR4A1 knockdown prevented viral propagation in culture, reduced LC3 levels, and increased LAMP2 expression. We therefore describe a drug-able pathway whereby MDM2 ubiquitinates and degrades KRT10, increasing NR4A1 expression and allowing VZV replication and propagation.


Assuntos
Regulação da Expressão Gênica , Herpes Zoster/genética , Herpes Zoster/metabolismo , Herpesvirus Humano 3/fisiologia , Queratina-10/genética , Queratinócitos/patologia , RNA/genética , Replicação Viral , Herpes Zoster/virologia , Humanos , Queratina-10/biossíntese , Queratinócitos/metabolismo , Queratinócitos/virologia
13.
Vaccine ; 38(2): 150-157, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31679866

RESUMO

Herpes zoster (HZ) is caused by reactivation of varicella zoster virus (VZV) that established latency in sensory and autonomic neurons during primary infection. In the Shingles Prevention Study (SPS), a large efficacy trial of live attenuated Oka/Merck zoster vaccine (ZVL), PCR-confirmed second episodes of HZ occurred in two of 660 placebo and one of 321 ZVL recipients with documented HZ during a mean follow-up of 3.13 years. An additional two ZVL recipients experienced a second episode of HZ in the Long-Term Persistence Substudy. All episodes of HZ were caused by wild-type VZV. The first and second episodes of HZ occurred in different dermatomes in each of these five participants, with contralateral recurrences in two. Time between first and second episodes ranged from 12 to 28 months. One of the five participants, who was immunocompetent on study enrollment, was immunocompromised at the onset of his first and second episodes of HZ. VZV DNA isolated from rash lesions from the first and second episodes of HZ was used to sequence the full-length VZV genomes. For the unique-sequence regions of the VZV genome, we employed target enrichment of VZV DNA, followed by deep sequencing. For the reiteration regions, we used PCR amplification and Sanger sequencing. Our analysis and comparison of the VZV genomes from the first and second episodes of HZ in each of the five participants indicate that both episodes were caused by the same VZV strain. This is consistent with the extraordinary stability of VZV during the replication phase of varicella and the subsequent establishment of latency in sensory ganglia throughout the body. Our observations also indicate that VZV is stable during the persistence of latency and the subsequent reactivation and replication that results in HZ.


Assuntos
Vacina contra Herpes Zoster/administração & dosagem , Herpes Zoster/virologia , Herpesvirus Humano 3/isolamento & purificação , Idoso , Feminino , Seguimentos , Herpes Zoster/imunologia , Herpes Zoster/prevenção & controle , Herpesvirus Humano 3/imunologia , Humanos , Hospedeiro Imunocomprometido , Masculino , Reação em Cadeia da Polimerase , Recidiva , Fatores de Tempo
14.
PLoS Pathog ; 15(11): e1008076, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31725813

RESUMO

During latent infections with herpes simplex virus 1 (HSV-1), viral transcription is restricted and the genomes are mostly maintained in silenced chromatin, whereas in lytically infected cells all viral genes are transcribed and the genomes are dynamically chromatinized. Histones in the viral chromatin bear markers of silenced chromatin at early times in lytic infection or of active transcription at later times. The virion protein VP16 activates transcription of the immediate-early (IE) genes by recruiting transcription activators and chromatin remodelers to their promoters. Two IE proteins, ICP0 and ICP4 which modulate chromatin epigenetics, then activate transcription of early and late genes. Although chromatin is involved in the mechanism of activation of HSV- transcription, its precise role is not entirely understood. In the cellular genome, chromatin dynamics often modulate transcription competence whereas promoter-specific transcription factors determine transcription activity. Here, biophysical fractionation of serially digested HSV-1 chromatin followed by short-read deep sequencing indicates that nuclear HSV-1 DNA has different biophysical properties than protein-free or encapsidated HSV-1 DNA. The entire HSV-1 genomes in infected cells were equally accessible. The accessibility of transcribed or non-transcribed genes under any given condition did not differ, and each gene was entirely sampled in both the most and least accessible chromatin. However, HSV-1 genomes fractionated differently under conditions of generalized or restricted transcription. Approximately 1/3 of the HSV-1 DNA including fully sampled genes resolved to the most accessible chromatin when HSV-1 transcription was active, but such enrichment was reduced to only 3% under conditions of restricted HSV-1 transcription. Short sequences of restricted accessibility separated genes with different transcription levels. Chromatin dynamics thus provide a first level of regulation on HSV-1 transcription, dictating the transcriptional competency of the genomes during lytic infections, whereas the transcription of individual genes is then most likely activated by specific transcription factors. Moreover, genes transcribed to different levels are separated by short sequences with limited accessibility.


Assuntos
Cromatina/metabolismo , Regulação Viral da Expressão Gênica , Genes Virais , Genoma Viral , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Replicação Viral , Animais , Chlorocebus aethiops , Cromatina/genética , Herpes Simples/genética , Humanos , Células Vero
15.
Mol Biol Evol ; 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31273385

RESUMO

Epstein-Barr virus (EBV) is one of the most common viral infections in humans and persists within its host for life. EBV therefore represents an extremely successful virus that has evolved complex strategies to evade the host's innate and adaptive immune response during both initial and persistent stages of infection. Here, we conducted a comparative genomics analysis on 223 whole genome sequences of world-wide EBV strains. We recover extensive genome-wide linkage disequilibrium (LD) despite pervasive genetic recombination. This pattern is explained by the global EBV population being subdivided into three main sub-populations, one primarily found in East Asia, one in Southeast Asia and Oceania, and the third including most of the other globally distributed genomes we analyzed. Additionally, sites in LD were overrepresented in immunogenic genes. Taken together, our results suggest that host immune selection and local adaptation to different human host populations has shaped the genome-wide patterns of genetic diversity in EBV.

16.
Proc Natl Acad Sci U S A ; 116(12): 5693-5698, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30819890

RESUMO

Recent sequencing efforts have led to estimates of human cytomegalovirus (HCMV) genome-wide intrahost diversity that rival those of persistent RNA viruses [Renzette N, Bhattacharjee B, Jensen JD, Gibson L, Kowalik TF (2011) PLoS Pathog 7:e1001344]. Here, we deep sequence HCMV genomes recovered from single and longitudinally collected blood samples from immunocompromised children to show that the observations of high within-host HCMV nucleotide diversity are explained by the frequent occurrence of mixed infections caused by genetically distant strains. To confirm this finding, we reconstructed within-host viral haplotypes from short-read sequence data. We verify that within-host HCMV nucleotide diversity in unmixed infections is no greater than that of other DNA viruses analyzed by the same sequencing and bioinformatic methods and considerably less than that of human immunodeficiency and hepatitis C viruses. By resolving individual viral haplotypes within patients, we reconstruct the timing, likely origins, and natural history of superinfecting strains. We uncover evidence for within-host recombination between genetically distinct HCMV strains, observing the loss of the parental virus containing the nonrecombinant fragment. The data suggest selection for strains containing the recombinant fragment, generating testable hypotheses about HCMV evolution and pathogenesis. These results highlight that high HCMV diversity present in some samples is caused by coinfection with multiple distinct strains and provide reassurance that within the host diversity for single-strain HCMV infections is no greater than for other herpesviruses.


Assuntos
Citomegalovirus/genética , Recombinação Genética/genética , Superinfecção/genética , Sequência de Bases/genética , Criança , Pré-Escolar , Infecções por Citomegalovirus/virologia , DNA Viral/genética , Feminino , Variação Genética/genética , Genoma Humano/genética , Genoma Viral , Haplótipos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Hospedeiro Imunocomprometido/genética , Lactente , Recém-Nascido , Masculino , Análise de Sequência de DNA/métodos
17.
Nat Commun ; 10(1): 754, 2019 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-30765700

RESUMO

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.


Assuntos
Genes Virais/genética , Herpesvirus Humano 1/genética , Nanoporos , Análise de Sequência de RNA/métodos , Transcriptoma/genética , Linhagem Celular , Células Cultivadas , Células Epiteliais/virologia , Fibroblastos/virologia , Genoma Viral/genética , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Neurônios/citologia , Neurônios/virologia , RNA Viral/genética , Proteínas Virais/genética
18.
J Virol ; 93(1)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30305358

RESUMO

Transcriptome profiling has become routine in studies of many biological processes. However, the favored approaches such as short-read Illumina RNA sequencing are giving way to long-read sequencing platforms better suited to interrogating the complex transcriptomes typical of many RNA and DNA viruses. Here, we provide a guide-tailored to molecular virologists-to the ins and outs of viral transcriptome sequencing and discuss the strengths and weaknesses of the major RNA sequencing technologies as tools to analyze the abundance and diversity of the viral transcripts made during infection.


Assuntos
Perfilação da Expressão Gênica/métodos , Vírus de RNA/genética , Análise de Sequência de RNA/instrumentação , Biologia Computacional/métodos , Perfilação da Expressão Gênica/instrumentação , Regulação Viral da Expressão Gênica , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Nanoporos , Análise de Sequência de RNA/métodos , Análise de Célula Única
19.
Genes Dev ; 32(23-24): 1472-1484, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30463905

RESUMO

Modification of mRNA by N 6-adenosine methylation (m6A) on internal bases influences gene expression in eukaryotes. How the dynamic genome-wide landscape of m6A-modified mRNAs impacts virus infection and host immune responses remains poorly understood. Here, we show that type I interferon (IFN) production triggered by dsDNA or human cytomegalovirus (HCMV) is controlled by the cellular m6A methyltrasferase subunit METTL14 and ALKBH5 demethylase. While METTL14 depletion reduced virus reproduction and stimulated dsDNA- or HCMV-induced IFNB1 mRNA accumulation, ALKBH5 depletion had the opposite effect. Depleting METTL14 increased both nascent IFNB1 mRNA production and stability in response to dsDNA. In contrast, ALKBH5 depletion reduced nascent IFNB1 mRNA production without detectably influencing IFN1B mRNA decay. Genome-wide transcriptome profiling following ALKBH5 depletion identified differentially expressed genes regulating antiviral immune responses, while METTL14 depletion altered pathways impacting metabolic reprogramming, stress responses, and aging. Finally, we determined that IFNB1 mRNA was m6A-modified within both the coding sequence and the 3' untranslated region (UTR). This establishes that the host m6A modification machinery controls IFNß production triggered by HCMV or dsDNA. Moreover, it demonstrates that responses to nonmicrobial dsDNA in uninfected cells, which shape host immunity and contribute to autoimmune disease, are regulated by enzymes controlling m6A epitranscriptomic changes.


Assuntos
DNA/imunologia , Regulação da Expressão Gênica/genética , Sistema Imunitário/enzimologia , Imunidade Inata/genética , Interferon beta/genética , Metiltransferases/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Citomegalovirus/imunologia , Perfilação da Expressão Gênica , Humanos , Interferon beta/metabolismo , Estabilidade de RNA/genética , Células Vero , Replicação Viral/genética
20.
J Neurovirol ; 24(6): 797-812, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30414047

RESUMO

Meeting Report on the 8th Annual Symposium of the Colorado Alphaherpesvirus Latency Society (CALS), held on May 16-19, 2018, in Vail, Colorado.


Assuntos
Alphaherpesvirinae/fisiologia , Infecções por Herpesviridae/virologia , Latência Viral/fisiologia , Colorado , Humanos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...