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1.
EMBO Rep ; : e53543, 2021 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-34842321

RESUMO

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for dissecting the complexity of normal and diseased tissues, enabling characterization of cell diversity and heterogeneous phenotypic states in unprecedented detail. However, this technology has been underutilized for exploring the interactions between the host cell and viral pathogens in latently infected cells. Herein, we use scRNA-seq and single-molecule sensitivity fluorescent in situ hybridization (smFISH) technologies to investigate host single-cell transcriptome changes upon the reactivation of a human neurotropic virus, herpes simplex virus-1 (HSV-1). We identify the stress sensor growth arrest and DNA damage-inducible 45 beta (Gadd45b) as a critical antiviral host factor that regulates HSV-1 reactivation events in a subpopulation of latently infected primary neurons. We show that distinct subcellular localization of Gadd45b correlates with the viral late gene expression program, as well as the expression of the viral transcription factor, ICP4. We propose that a hallmark of a "successful" or "aborted" HSV-1 reactivation state in primary neurons is determined by a unique subcellular localization signature of the stress sensor Gadd45b.

2.
Proc Natl Acad Sci U S A ; 118(45)2021 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-34725147

RESUMO

In addition to being required for protein synthesis, ribosomes and ribosomal proteins (RPs) also regulate messenger RNA translation in uninfected and virus-infected cells. By individually depleting 85 RPs using RNA interference, we found that overall protein synthesis in uninfected primary fibroblasts was more sensitive to RP depletion than those infected with herpes simplex virus-1 (HSV-1). Although representative RP depletion (uL3, uS4, uL5) inhibited protein synthesis in cells infected with two different DNA viruses (human cytomegalovirus, vaccinia virus), HSV-1-infected cell protein synthesis unexpectedly endured and required a single virus-encoded gene product, VP22. During individual RP insufficiency, VP22-expressing HSV-1 replicated better than a VP22-deficient variant. Furthermore, VP22 promotes polysome accumulation in virus-infected cells when uL3 or ribosome availability is limiting and cosediments with initiating and elongating ribosomes in infected and uninfected cells. This identifies VP22 as a virus-encoded, ribosome-associated protein that compensates for RP insufficiency to support viral protein synthesis and replication. Moreover, it reveals an unanticipated class of virus-encoded, ribosome-associated effectors that reduce the dependence of protein synthesis upon host RPs and broadly support translation during physiological stress such as infection.

3.
J Thromb Haemost ; 19(12): 3139-3153, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34538015

RESUMO

OBJECTIVE: Heightened inflammation, dysregulated immunity, and thrombotic events are characteristic of hospitalized COVID-19 patients. Given that platelets are key regulators of thrombosis, inflammation, and immunity they represent prime candidates as mediators of COVID-19-associated pathogenesis. The objective of this study was to understand the contribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the platelet phenotype via phenotypic (activation, aggregation) and transcriptomic characterization. APPROACH AND RESULTS: In a cohort of 3915 hospitalized COVID-19 patients, we analyzed blood platelet indices collected at hospital admission. Following adjustment for demographics, clinical risk factors, medication, and biomarkers of inflammation and thrombosis, we find platelet count, size, and immaturity are associated with increased critical illness and all-cause mortality. Bone marrow, lung tissue, and blood from COVID-19 patients revealed the presence of SARS-CoV-2 virions in megakaryocytes and platelets. Characterization of COVID-19 platelets found them to be hyperreactive (increased aggregation, and expression of P-selectin and CD40) and to have a distinct transcriptomic profile characteristic of prothrombotic large and immature platelets. In vitro mechanistic studies highlight that the interaction of SARS-CoV-2 with megakaryocytes alters the platelet transcriptome, and its effects are distinct from the coronavirus responsible for the common cold (CoV-OC43). CONCLUSIONS: Platelet count, size, and maturity associate with increased critical illness and all-cause mortality among hospitalized COVID-19 patients. Profiling tissues and blood from COVID-19 patients revealed that SARS-CoV-2 virions enter megakaryocytes and platelets and associate with alterations to the platelet transcriptome and activation profile.


Assuntos
COVID-19 , Trombose , Plaquetas , Humanos , SARS-CoV-2 , Índice de Gravidade de Doença
5.
Proc Natl Acad Sci U S A ; 118(30)2021 07 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.

6.
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
7.
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
8.
Elife ; 82019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31841110

RESUMO

Ribosomes are universally important in biology and their production is dysregulated by developmental disorders, cancer, and virus infection. Although presumed required for protein synthesis, how ribosome biogenesis impacts virus reproduction and cell-intrinsic immune responses remains untested. Surprisingly, we find that restricting ribosome biogenesis stimulated human cytomegalovirus (HCMV) replication without suppressing translation. Interfering with ribosomal RNA (rRNA) accumulation triggered nucleolar stress and repressed expression of 1392 genes, including High Mobility Group Box 2 (HMGB2), a chromatin-associated protein that facilitates cytoplasmic double-stranded (ds) DNA-sensing by cGAS. Furthermore, it reduced cytoplasmic HMGB2 abundance and impaired induction of interferon beta (IFNB1) mRNA, which encodes a critical anti-proliferative, proinflammatory cytokine, in response to HCMV or dsDNA in uninfected cells. This establishes that rRNA accumulation regulates innate immune responses to dsDNA by controlling HMGB2 abundance. Moreover, it reveals that rRNA accumulation and/or nucleolar activity unexpectedly regulate dsDNA-sensing to restrict virus reproduction and regulate inflammation. (145 words).


Assuntos
DNA , Imunidade Inata , Ribossomos/metabolismo , Viroses , Linhagem Celular , Citocinas/metabolismo , Citomegalovirus/imunologia , Fibroblastos , Proteína HMGB2/genética , Humanos , Imunidade Inata/genética , Interferon beta , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , RNA Polimerase I , RNA Mensageiro/metabolismo , RNA Ribossômico , Viroses/genética , Replicação Viral
9.
Artigo em Inglês | MEDLINE | ID: mdl-31843932

RESUMO

Autophagy is a powerful host defense that restricts herpes simplex virus-1 (HSV-1) pathogenesis in neurons. As a countermeasure, the viral ICP34.5 polypeptide, which is exclusively encoded by HSV, antagonizes autophagy in part through binding Beclin1. However, whether autophagy is a cell-type-specific antiviral defense or broadly restricts HSV-1 reproduction in nonneuronal cells is unknown. Here, we establish that autophagy limits HSV-1 productive growth in nonneuronal cells and is repressed by the Us3 gene product. Phosphorylation of the autophagy regulators ULK1 and Beclin1 in virus-infected cells was dependent upon the HSV-1 Us3 Ser/Thr kinase. Furthermore, Beclin1 was unexpectedly identified as a direct Us3 kinase substrate. Although disabling autophagy did not impact replication of an ICP34.5-deficient virus in primary human fibroblasts, depleting Beclin1 and ULK1 partially rescued Us3-deficient HSV-1 replication. This shows that autophagy restricts HSV-1 reproduction in a cell-intrinsic manner in nonneuronal cells and is suppressed by multiple, independent viral functions targeting Beclin1 and ULK1. Moreover, it defines a surprising role regulating autophagy for the Us3 kinase, which unlike ICP34.5 is widely encoded by alpha-herpesvirus subfamily members.

10.
Proc Natl Acad Sci U S A ; 116(45): 22583-22590, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31636182

RESUMO

Gene expression is rapidly remodeled by infection and inflammation in part via transcription factor NF-κB activation and regulated protein synthesis. While protein synthesis is largely controlled by mRNA translation initiation, whether cellular translation elongation factors are responsive to inflammation and infection remains poorly understood. Here, we reveal a surprising mechanism whereby NF-κB restricts phosphorylation of the critical translation elongation factor eEF2, which catalyzes the protein synthesis translocation step. Upon exposure to NF-κB-activating stimuli, including TNFα, human cytomegalovirus infection, or double-stranded DNA, eEF2 phosphorylation on Thr56, which slows elongation to limit protein synthesis, and the overall abundance of eEF2 kinase (eEF2K) are reduced. Significantly, this reflected a p65 NF-κB subunit-dependent reduction in eEF2K pre-mRNA, indicating that NF-κB activation represses eEF2K transcription to decrease eEF2K protein levels. Finally, we demonstrate that reducing eEF2K abundance regulates protein synthesis in response to a bacterial toxin that inactivates eEF2. This establishes that NF-κB activation by diverse physiological effectors controls eEF2 activity via a transcriptional repression mechanism that reduces eEF2K polypeptide abundance to preclude eEF2 phosphorylation, thereby stimulating translation elongation and protein synthesis. Moreover, it illustrates how nuclear transcription regulation shapes translation elongation factor activity and exposes how eEF2 is integrated into innate immune response networks orchestrated by NF-κB.


Assuntos
DNA/metabolismo , Quinase do Fator 2 de Elongação/genética , Inflamação/metabolismo , Biossíntese de Proteínas , Fator de Transcrição RelA/metabolismo , Motivos de Aminoácidos , DNA/genética , Quinase do Fator 2 de Elongação/química , Quinase do Fator 2 de Elongação/metabolismo , Humanos , Inflamação/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Fator 2 de Elongação de Peptídeos/genética , Fator 2 de Elongação de Peptídeos/metabolismo , Fosforilação , Fator de Transcrição RelA/genética
11.
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
12.
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
13.
Artigo em Inglês | MEDLINE | ID: mdl-29891561

RESUMO

As obligate intracellular parasites, virus reproduction requires host cell functions. Despite variations in genome size and configuration, nucleic acid composition, and their repertoire of encoded functions, all viruses remain unconditionally dependent on the protein synthesis machinery resident within their cellular hosts to translate viral messenger RNAs (mRNAs). A complex signaling network responsive to physiological stress, including infection, regulates host translation factors and ribosome availability. Furthermore, access to the translation apparatus is patrolled by powerful host immune defenses programmed to restrict viral invaders. Here, we review the tactics and mechanisms used by viruses to appropriate control over host ribosomes, subvert host defenses, and dominate the infected cell translational landscape. These not only define aspects of infection biology paramount for virus reproduction, but continue to drive fundamental discoveries into how cellular protein synthesis is controlled in health and disease.


Assuntos
Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Biossíntese de Proteínas , Animais , Humanos , Vírus de Plantas/metabolismo , Processamento Pós-Transcricional do RNA , Ribossomos/metabolismo , Estresse Fisiológico , Proteínas Virais/biossíntese , Viroses/metabolismo , Replicação Viral
14.
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
15.
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
16.
J Virol ; 92(24)2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30282708

RESUMO

By sensing fundamental parameters, including nutrient availability, activated mechanistic target of rapamycin complex 1 (mTORC1) suppresses catabolic outcomes and promotes anabolic processes needed for herpes simplex virus 1 (HSV-1) productive growth. While the virus-encoded Us3 Ser/Thr kinase is required to activate mTORC1, whether stress associated with amino acid insufficiency impacts mTORC1 activation in infected cells and virus reproduction was unknown. In contrast to uninfected cells, where amino acid withdrawal inhibits mTORC1 activation, we demonstrate that mTORC1 activity is sustained in HSV-1-infected cells during amino acid insufficiency. We show that in the absence of Us3, the insensitivity of mTORC1 to amino acid withdrawal in infected cells was dependent on the host kinase Akt and establish a role for the HSV-1 UL46 gene product, which stimulates phosphatidylinositol (PI) 3-kinase signaling. Significantly, virus reproduction during amino acid insufficiency was stimulated by the viral UL46 gene product. By synergizing with Us3, UL46 reprograms mTORC1 such that it is insensitive to amino acid withdrawal and supports sustained mTORC1 activation and virus reproduction during amino acid insufficiency. This identifies an unexpected function for UL46 in supporting virus reproduction during physiological stress and identifies a new class of virus-encoded mTORC1 regulators that selectively uncouple mTORC1 activation from amino acid sufficiency.IMPORTANCE Mechanistic target of rapamycin complex 1 (mTORC1) is a multisubunit cellular kinase that coordinates protein synthesis with changing amino acid levels. During amino acid insufficiency, mTORC1 is repressed in uninfected cells, dampening protein synthesis and potentially restricting virus reproduction. Here, we establish that HSV-1 alters the responsiveness of mTORC1 to metabolic stress resulting from amino acid insufficiency. Unlike in uninfected cells, mTORC1 remains activated in HSV-1-infected cells deprived of amino acids. Synergistic action of the HSV-1 UL46 gene product, which stimulates PI 3-kinase, and the Us3 kinase supports virus reproduction during amino acid withdrawal. These results define how HSV-1, a medically important human pathogen associated with a range of diseases, uncouples mTORC1 activation from amino acid availability. Furthermore, they help explain how the virus reproduces during physiological stress. Reproduction triggered by physiological stress is characteristic of herpesvirus infections, where lifelong latency is punctuated by episodic reactivation events.


Assuntos
Aminoácidos/deficiência , Antígenos Virais/metabolismo , Herpesvirus Humano 1/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Regulação Viral da Expressão Gênica , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Vero , Replicação Viral
17.
Mol Ther Oncolytics ; 8: 71-81, 2018 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-29888320

RESUMO

Through the action of two virus-encoded decapping enzymes (D9 and D10) that remove protective caps from mRNA 5'-termini, Vaccinia virus (VACV) accelerates mRNA decay and limits activation of host defenses. D9- or D10-deficient VACV are markedly attenuated in mice and fail to counter cellular double-stranded RNA-responsive innate immune effectors, including PKR. Here, we capitalize upon this phenotype and demonstrate that VACV deficient in either decapping enzyme are effective oncolytic viruses. Significantly, D9- or D10-deficient VACV displayed anti-tumor activity against syngeneic mouse tumors of different genetic backgrounds and human hepatocellular carcinoma xenografts. Furthermore, D9- and D10-deficient VACV hyperactivated the host anti-viral enzyme PKR in non-tumorigenic cells compared to wild-type virus. This establishes a new genetic platform for oncolytic VACV development that is deficient for a major pathogenesis determinant while retaining viral genes that support robust productive replication like those required for nucleotide metabolism. It further demonstrates how VACV mutants unable to execute a fundamental step in virus-induced mRNA decay can be unexpectedly translated into a powerful anti-tumor therapy.

18.
J Virol ; 92(15)2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29793959

RESUMO

In response to virus-induced shutoff host protein synthesis, dynamic aggregates containing mRNA, RNA-binding proteins and translation factors termed stress granules (SGs) often accumulate within the cytoplasm. SGs typically form following phosphorylation and inactivation of the eukaryotic translation initiation factor 2α (eIF2α), a substrate of the double-stranded RNA (dsRNA)-activated kinase protein kinase R (PKR). The detection of innate immune sensors and effectors like PKR at SGs suggests a role in pathogen nucleic acid sensing. However, the functional importance of SGs in host innate responses is unclear and has primarily been examined in response to infection with select RNA viruses. During infection with the DNA virus herpes simplex virus 1 (HSV-1), the virus-encoded virion host shutoff (VHS) endoribonuclease is required to restrict interferon production, PKR activation, and SG formation, although the relationship between these activities remains incompletely understood. Here, we show that in cells infected with a VHS-deficient HSV-1 (ΔVHS) dsRNA accumulated and localized to SGs. Surprisingly, formation of dsRNA and its concentration at SGs was not required for beta interferon mRNA induction, indicating that suppression of type I interferon induction by VHS does not stem from its control of dsRNA accumulation. Instead, STING signaling downstream of cGMP-AMP synthase (cGAS)-dependent DNA sensing is required for beta interferon induction. In contrast, significantly less PKR activation is observed when SG assembly is disrupted by ISRIB, an inhibitor of phosphorylated eIF2α-mediated translation repression, or depleting SG scaffolding proteins G3BP1 or TIA1. This demonstrates that PKR activation is intimately linked to SG formation and that SGs form important hubs to potentiate PKR activation during infection.IMPORTANCE Formation of cytoplasmic stress granules that are enriched for innate immune sensors and effectors is suppressed during many viral infections. It is unclear, however, to what extent this is a side effect of viral efforts to maintain protein synthesis or intentional disruption of a hub for innate immune sensing. In this study, we utilize a herpes simplex virus 1 mutant lacking the RNA nuclease VHS which upon infection induces SGs, PKR activation, and beta interferon to address this question. We show that dsRNA is localized to SGs and that SGs can function to promote PKR activation in the context of a DNA virus infection, but we find no evidence to support their importance for interferon induction during HSV-1 infection.


Assuntos
Grânulos Citoplasmáticos/imunologia , Fibroblastos/imunologia , Herpesvirus Humano 1/imunologia , Imunidade Inata , Ribonucleases/imunologia , Transdução de Sinais/imunologia , Proteínas Virais/imunologia , Células Cultivadas , Grânulos Citoplasmáticos/enzimologia , Grânulos Citoplasmáticos/genética , Fibroblastos/metabolismo , Fibroblastos/virologia , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Nucleotidiltransferases/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/imunologia , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , RNA Viral/imunologia , RNA Viral/metabolismo , Ribonucleases/genética , Ribonucleases/metabolismo , Transdução de Sinais/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo
19.
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
20.
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.

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