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1.
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.

2.
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
3.
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
4.
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
5.
Methods Mol Biol ; 2060: 263-277, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31617183

RESUMO

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-Dawley
6.
J Neurovirol ; 26(2): 297-309, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31502208

RESUMO

Meeting Report on the 9th Annual Symposium of the Colorado Alphaherpesvirus Latency Society (CALS) held on May 8-11, 2019, in Vail, CO.


Assuntos
Alphaherpesvirinae/fisiologia , Infecções por Herpesviridae/virologia , Latência Viral , Colorado , Humanos , Sociedades Médicas
7.
Mol Cell ; 74(3): 466-480.e4, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30930055

RESUMO

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/genética
8.
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
9.
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
10.
Virology ; 512: 124-131, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28957690

RESUMO

Herpes simplex virus 1 (HSV-1) is a widespread pathogen that persists for life, replicating in surface tissues and establishing latency in peripheral ganglia. Increasingly, molecular studies of latency use cultured neuron models developed using recombinant viruses such as HSV-1 GFP-US11, a derivative of strain Patton expressing green fluorescent protein (GFP) fused to the viral US11 protein. Visible fluorescence follows viral DNA replication, providing a real time indicator of productive infection and reactivation. Patton was isolated in Houston, Texas, prior to 1973, and distributed to many laboratories. Although used extensively, the genomic structure and phylogenetic relationship to other strains is poorly known. We report that wild type Patton and the GFP-US11 recombinant contain the full complement of HSV-1 genes and differ within the unique regions at only eight nucleotides, changing only two amino acids. Although isolated in North America, Patton is most closely related to Asian viruses, including KOS63.


Assuntos
Herpes Simples/virologia , Herpesvirus Humano 1/genética , Ásia/epidemiologia , Sequência Conservada , DNA Viral , Regulação Viral da Expressão Gênica , Herpes Simples/epidemiologia , Humanos , Filogenia , Replicação Viral
11.
Viruses ; 9(8)2017 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-28783105

RESUMO

Infection with herpes simplex virus-1 (HSV-1) brings numerous changes in cellular gene expression. Levels of most host mRNAs are reduced, limiting synthesis of host proteins, especially those involved in antiviral defenses. The impact of HSV-1 on host microRNAs (miRNAs), an extensive network of short non-coding RNAs that regulate mRNA stability/translation, remains largely unexplored. Here we show that transcription of the miR-183 cluster (miR-183, miR-96, and miR-182) is selectively induced by HSV-1 during productive infection of primary fibroblasts and neurons. ICP0, a viral E3 ubiquitin ligase expressed as an immediate-early protein, is both necessary and sufficient for this induction. Nuclear exclusion of ICP0 or removal of the RING (really interesting new gene) finger domain that is required for E3 ligase activity prevents induction. ICP0 promotes the degradation of numerous host proteins and for the most part, the downstream consequences are unknown. Induction of the miR-183 cluster can be mimicked by depletion of host transcriptional repressors zinc finger E-box binding homeobox 1 (ZEB1)/-crystallin enhancer binding factor 1 (δEF1) and zinc finger E-box binding homeobox 2 (ZEB2)/Smad-interacting protein 1 (SIP1), which we establish as new substrates for ICP0-mediated degradation. Thus, HSV-1 selectively stimulates expression of the miR-183 cluster by ICP0-mediated degradation of ZEB transcriptional repressors.


Assuntos
Herpesvirus Humano 1/enzimologia , Interações Hospedeiro-Patógeno , MicroRNAs/genética , Ubiquitina-Proteína Ligases/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Núcleo Celular , Células Cultivadas , Fibroblastos/virologia , Regulação da Expressão Gênica , Herpesvirus Humano 1/genética , Humanos , Proteínas Imediatamente Precoces/deficiência , Proteínas Imediatamente Precoces/genética , Proteínas do Tecido Nervoso/genética , Neurônios/virologia , Ligação Proteica , Proteólise , Proteínas de Ligação a RNA/genética , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Replicação Viral , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética
12.
Pathogens ; 6(2)2017 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-28594343

RESUMO

Herpes simplex virus 1 (HSV-1) uses latency in peripheral ganglia to persist in its human host, however, recurrent reactivation from this reservoir can cause debilitating and potentially life-threatening disease. Most studies of latency use live-animal infection models, but these are complex, multilayered systems and can be difficult to manipulate. Infection of cultured primary neurons provides a powerful alternative, yielding important insights into host signaling pathways controlling latency. However, small animal models do not recapitulate all aspects of HSV-1 infection in humans and are limited in terms of the available molecular tools. To address this, we have developed a latency model based on human neurons differentiated in culture from an NIH-approved embryonic stem cell line. The resulting neurons are highly permissive for replication of wild-type HSV-1, but establish a non-productive infection state resembling latency when infected at low viral doses in the presence of the antivirals acyclovir and interferon-α. In this state, viral replication and expression of a late viral gene marker are not detected but there is an accumulation of the viral latency-associated transcript (LAT) RNA. After a six-day establishment period, antivirals can be removed and the infected cultures maintained for several weeks. Subsequent treatment with sodium butyrate induces reactivation and production of new infectious virus. Human neurons derived from stem cells provide the appropriate species context to study this exclusively human virus with the potential for more extensive manipulation of the progenitors and access to a wide range of preexisting molecular tools.

13.
Cell Rep ; 18(5): 1312-1323, 2017 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-28147283

RESUMO

How type I and II interferons prevent periodic reemergence of latent pathogens in tissues of diverse cell types remains unknown. Using homogeneous neuron cultures latently infected with herpes simplex virus 1, we show that extrinsic type I or II interferon acts directly on neurons to induce unique gene expression signatures and inhibit the reactivation-specific burst of viral genome-wide transcription called phase I. Surprisingly, interferons suppressed reactivation only during a limited period early in phase I preceding productive virus growth. Sensitivity to type II interferon was selectively lost if viral ICP0, which normally accumulates later in phase I, was expressed before reactivation. Thus, interferons suppress reactivation by preventing initial expression of latent genomes but are ineffective once phase I viral proteins accumulate, limiting interferon action. This demonstrates that inducible reactivation from latency is only transiently sensitive to interferon. Moreover, it illustrates how latent pathogens escape host immune control to periodically replicate by rapidly deploying an interferon-resistant state.


Assuntos
Replicação do DNA/imunologia , Expressão Gênica/imunologia , Latência Viral/imunologia , Replicação Viral/imunologia , Animais , Células Cultivadas , Genoma Viral/imunologia , Herpesvirus Humano 1/imunologia , Interferon Tipo I/imunologia , Interferon gama/imunologia , Neurônios/imunologia , Neurônios/virologia , Ratos , Ratos Sprague-Dawley , Transcrição Genética/imunologia , Proteínas Virais/imunologia , Ativação Viral/imunologia
14.
J Virol ; 91(2)2017 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-27807236

RESUMO

Herpes simplex virus (HSV) establishes a latent reservoir in neurons of human peripheral nerves. In this quiescent state, the viral genome persists as a circular, histone-associated episome, and transcription of viral lytic cycle genes is largely suppressed through epigenetic processes. Periodically, latent virus undergoes reactivation whereby lytic genes are activated and viral replication occurs. In this Gem, we review recent evidence that mechanisms governing the initial transcription of lytic genes are distinct from those of de novo infection and directly link reactivation to neuronal stress response pathways. We also discuss evidence that lytic cycle gene expression can be uncoupled from the full reactivation program, arguing for a less sharply bimodal definition of latency.


Assuntos
Regulação Viral da Expressão Gênica , Herpes Simples/virologia , Simplexvirus/fisiologia , Transcrição Genética , Ativação Viral , Replicação Viral , Animais , Herpes Simples/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Neurônios/metabolismo , Neurônios/virologia , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico , Latência Viral
15.
Methods Mol Biol ; 1144: 167-79, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24671683

RESUMO

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 and 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 it 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
Herpesvirus Humano 1/efeitos dos fármacos , Biologia Molecular/métodos , Ativação Viral/genética , Latência Viral/genética , Animais , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/crescimento & desenvolvimento , Humanos , Neurônios/citologia , Cultura Primária de Células
16.
J Virol ; 88(4): 2337-9, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24307587

RESUMO

To facilitate studies of herpes simplex virus 1 latency, cell culture models of quiescent or latent infection have been developed. Using deep sequencing, we analyzed the expression of viral microRNAs (miRNAs) in two models employing human fibroblasts and one using rat neurons. In all cases, the expression patterns differed from that in productively infected cells, with the rat neuron pattern most closely resembling that found in latently infected human or mouse ganglia in vivo.


Assuntos
Herpes Simples/metabolismo , Herpesvirus Humano 1/genética , MicroRNAs/metabolismo , Latência Viral/genética , Animais , Técnicas de Cultura de Células , Fibroblastos/metabolismo , Herpes Simples/genética , Herpesvirus Humano 1/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Neurônios/metabolismo , Ratos
17.
Trends Microbiol ; 20(12): 604-11, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22963857

RESUMO

After replicating in surface epithelia, herpes simplex virus type-1 (HSV-1) enters the axonal terminals of peripheral neurons. The viral genome translocates to the nucleus, where it establishes a specialized infection known as latency, re-emerging periodically to seed new infections. Studies using cultured neuron models that faithfully recapitulate the molecular hallmarks of latency and reactivation defined in live animal models have provided fresh insight into the control of latency and connections to neuronal physiology. With this comes a growing appreciation for how the life cycles of HSV-1 and other herpesviruses are governed by key host pathways controlling metabolic homeostasis and cell identity.


Assuntos
Herpesvirus Humano 1/fisiologia , Neurônios/virologia , Ativação Viral , Latência Viral , Animais , Herpesvirus Humano 1/patogenicidade , Humanos
18.
Genes Dev ; 26(14): 1527-32, 2012 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-22802527

RESUMO

Latent herpes simplex virus-1 (HSV1) genomes in peripheral nerve ganglia periodically reactivate, initiating a gene expression program required for productive replication. Whether molecular cues detected by axons can be relayed to cell bodies and harnessed to regulate latent genome expression in neuronal nuclei is unknown. Using a neuron culture model, we found that inhibiting mTOR, depleting its regulatory subunit raptor, or inducing hypoxia all trigger reactivation. While persistent mTORC1 activation suppressed reactivation, a mutant 4E-BP (eIF4E-binding protein) translational repressor unresponsive to mTORC1 stimulated reactivation. Finally, inhibiting mTOR in axons induced reactivation. Thus, local changes in axonal mTOR signaling that control translation regulate latent HSV1 genomes in a spatially segregated compartment.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Axônios/metabolismo , Herpes Simples/metabolismo , Herpesvirus Humano 1/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Latência Viral/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Axônios/virologia , Células Cultivadas , Genoma Viral/fisiologia , Herpes Simples/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos , Proteínas/genética , Proteínas/metabolismo , Ratos , Serina-Treonina Quinases TOR/genética
19.
J Vis Exp ; (62)2012 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-22491318

RESUMO

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility. Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA(+) neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor(1). A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.


Assuntos
Técnicas de Cultura de Células/métodos , Herpesvirus Humano 1/fisiologia , Neurônios/citologia , Neurônios/virologia , Animais , Herpesvirus Humano 1/genética , Ratos , Recombinação Genética , Gânglio Cervical Superior/citologia , Virologia/métodos , Ativação Viral/fisiologia , Latência Viral
20.
PLoS Pathog ; 8(2): e1002540, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22383875

RESUMO

Herpes simplex virus type-1 (HSV-1) establishes latency in peripheral neurons, creating a permanent source of recurrent infections. The latent genome is assembled into chromatin and lytic cycle genes are silenced. Processes that orchestrate reentry into productive replication (reactivation) remain poorly understood. We have used latently infected cultures of primary superior cervical ganglion (SCG) sympathetic neurons to profile viral gene expression following a defined reactivation stimulus. Lytic genes are transcribed in two distinct phases, differing in their reliance on protein synthesis, viral DNA replication and the essential initiator protein VP16. The first phase does not require viral proteins and has the appearance of a transient, widespread de-repression of the previously silent lytic genes. This allows synthesis of viral regulatory proteins including VP16, which accumulate in the cytoplasm of the host neuron. During the second phase, VP16 and its cellular cofactor HCF-1, which is also predominantly cytoplasmic, concentrate in the nucleus where they assemble an activator complex on viral promoters. The transactivation function supplied by VP16 promotes increased viral lytic gene transcription leading to the onset of genome amplification and the production of infectious viral particles. Thus regulated localization of de novo synthesized VP16 is likely to be a critical determinant of HSV-1 reactivation in sympathetic neurons.


Assuntos
Inativação Gênica , Proteína Vmw65 do Vírus do Herpes Simples/fisiologia , Herpesvirus Humano 1/fisiologia , Neurônios/virologia , Plasmídeos/genética , Transcrição Genética , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Células Cultivadas , Inativação Gênica/fisiologia , Proteína Vmw65 do Vírus do Herpes Simples/metabolismo , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Homologia de Sequência , Fatores de Tempo , Transcrição Genética/genética , Latência Viral/genética , Latência Viral/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologia
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