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1.
Nat Commun ; 12(1): 1379, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33654074

RESUMO

Many immune responses depend upon activation of NF-κB, an important transcription factor in the elicitation of a cytokine response. Here we show that N4BP1 inhibits TLR-dependent activation of NF-κB by interacting with the NF-κB signaling essential modulator (NEMO, also known as IκB kinase γ) to attenuate NEMO-NEMO dimerization or oligomerization. The UBA-like (ubiquitin associated-like) and CUE-like (ubiquitin conjugation to ER degradation-like) domains in N4BP1 mediate interaction with the NEMO COZI domain. Both in vitro and in mice, N4bp1 deficiency specifically enhances TRIF-independent (TLR2, TLR7, or TLR9-mediated) but not TRIF-dependent (TLR3 or TLR4-mediated) NF-κB activation, leading to increased production of proinflammatory cytokines. In response to TLR4 or TLR3 activation, TRIF causes activation of caspase-8, which cleaves N4BP1 distal to residues D424 and D490 and abolishes its inhibitory effect. N4bp1-/- mice also have diminished numbers of T cells in the peripheral blood. Our work identifies N4BP1 as an inhibitory checkpoint protein that must be overcome to activate NF-κB, and a TRIF-initiated caspase-8-dependent mechanism by which this is accomplished.


Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Multimerização Proteica , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Caspase 8/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Herpesvirus Humano 1/fisiologia , Humanos , Interleucina-6/sangue , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos Endogâmicos C57BL , Mutação/genética , Inibidor de NF-kappaB alfa/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Multimerização Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina/metabolismo
2.
Viruses ; 13(2)2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33525505

RESUMO

The herpes simplex virus 1 (HSV-1) genome is extremely rich in guanine tracts that fold into G-quadruplexes (G4s), nucleic acid secondary structures implicated in key biological functions. Viral G4s were visualized in HSV-1 infected cells, with massive virus cycle-dependent G4-formation peaking during viral DNA replication. Small molecules that specifically interact with G4s have been shown to inhibit HSV-1 DNA replication. We here investigated the antiviral activity of TMPyP4, a porphyrin known to interact with G4s. The analogue TMPyP2, with lower G4 affinity, was used as control. We showed by biophysical analysis that TMPyP4 interacts with HSV-1 G4s, and inhibits polymerase progression in vitro; in infected cells, it displayed good antiviral activity which, however, was independent of inhibition of virus DNA replication or entry. At low TMPyP4 concentration, the virus released by the cells was almost null, while inside the cell virus amounts were at control levels. TEM analysis showed that virus particles were trapped inside cytoplasmatic vesicles, which could not be ascribed to autophagy, as proven by RT-qPCR, western blot, and immunofluorescence analysis. Our data indicate a unique mechanism of action of TMPyP4 against HSV-1, and suggest the unprecedented involvement of currently unknown G4s in viral or antiviral cellular defense pathways.


Assuntos
Antivirais/farmacologia , Quadruplex G/efeitos dos fármacos , Herpesvirus Humano 1/efeitos dos fármacos , Porfirinas/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/metabolismo , DNA Viral/química , DNA Viral/efeitos dos fármacos , Herpesvirus Humano 1/fisiologia , Ligantes , Estrutura Molecular , Porfirinas/química , Células Vero , Vírion/efeitos dos fármacos , Vírion/metabolismo
3.
Nucleic Acids Res ; 49(4): 2044-2064, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33533922

RESUMO

The integrity and regulation of the nuclear lamina is essential for nuclear organization and chromatin stability, with its dysregulation being linked to laminopathy diseases and cancer. Although numerous posttranslational modifications have been identified on lamins, few have been ascribed a regulatory function. Here, we establish that lamin B1 (LMNB1) acetylation at K134 is a molecular toggle that controls nuclear periphery stability, cell cycle progression, and DNA repair. LMNB1 acetylation prevents lamina disruption during herpesvirus type 1 (HSV-1) infection, thereby inhibiting virus production. We also demonstrate the broad impact of this site on laminar processes in uninfected cells. LMNB1 acetylation negatively regulates canonical nonhomologous end joining by impairing the recruitment of 53BP1 to damaged DNA. This defect causes a delay in DNA damage resolution and a persistent activation of the G1/S checkpoint. Altogether, we reveal LMNB1 acetylation as a mechanism for controlling DNA repair pathway choice and stabilizing the nuclear periphery.


Assuntos
Reparo do DNA , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Lamina Tipo B/metabolismo , Acetilação , Linhagem Celular , Núcleo Celular/virologia , Cromatina/metabolismo , Dano ao DNA , Feminino , Herpesvirus Humano 1/fisiologia , Humanos , Lamina Tipo B/química , Lisina/metabolismo , Lâmina Nuclear/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
5.
Viruses ; 13(1)2021 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-33435520

RESUMO

Herpes simplex virus type 1 (HSV-1) causes a lifelong latent infection with an estimated global prevalence of 66%. Primary and recurrent HSV infections are characterized by a tingling sensation, followed by an eruption of vesicles, which can cause painful erosions. Commonly used antiviral drugs against HSV infection are nucleoside analogues including acyclovir (ACV), famciclovir, and valacyclovir. Although these nucleoside analogues reduce morbidity and mortality in immunocompetent individuals, ACV-resistant HSV strains (ACVR-HSV) have been isolated from immunocompromised patients. Thus, ACVR-HSV infection poses a critical emerging public health concern. Recently, we reported that ginkgolic acid (GA) inhibits HSV-1 by disrupting viral structure, blocking fusion, and inhibiting viral protein synthesis. Additionally, we showed GA affords a broad spectrum of fusion inhibition of all three classes of fusion proteins, including those of HIV, Ebola, influenza A and Epstein Barr viruses. Here we report GA's antiviral activity against HSV-1 skin infection in BALB/cJ mice. GA-treated mice demonstrated a significantly reduced mortality rate and decreased infection scores compared to controls treated with dimethylsulfoxide (DMSO)-vehicle. Furthermore, GA efficiently inhibited ACVR-HSV-1 strain 17+ in vitro and in vivo. Since GA's mechanism of action includes virucidal activity and fusion inhibition, it is expected to work alone or synergistically with other anti-viral drugs, and we anticipate it to be effective against additional cutaneous and potentially systemic viral infections.


Assuntos
Antivirais/farmacologia , Dermatite/virologia , Herpes Simples/virologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/fisiologia , Salicilatos/farmacologia , Animais , Linhagem Celular , Chlorocebus aethiops , Dermatite/tratamento farmacológico , Modelos Animais de Doenças , Herpes Simples/tratamento farmacológico , Herpes Simples/transmissão , Camundongos , Células Vero , Ensaio de Placa Viral , Replicação Viral/efeitos dos fármacos
6.
J Virol ; 95(6)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33361424

RESUMO

Extracellular vesicles (EVs) are released by all types of cells as a means of intercellular communication. Their significance lies in the fact that they can alter recipient cell functions, despite their limited capacity for cargo. We have previously demonstrated that herpes simplex virus 1 (HSV-1) infection influences the cargo and functions of EVs released by infected cells and that these EVs negatively impact a subsequent HSV-1 infection. In the present study, we have implemented cutting-edge technologies to further characterize EVs released during HSV-1 infection. We identified distinct EV populations that were separable through a gradient approach. One population was positive for the tetraspanin CD63 and was distinct from EVs carrying components of the endosomal sorting complexes required for transport (ESCRT). Nanoparticle tracking analysis (NTA) combined with protein analysis indicated that the production of CD63+ EVs was selectively induced upon HSV-1 infection. The ExoView platform supported these data and suggested that the amount of CD63 per vesicle is larger upon infection. This platform also identified EV populations positive for other tetraspanins, including CD81 and CD9, whose abundance decreased upon HSV-1 infection. The stimulator of interferon genes (STING) was found in CD63+ EVs released during HSV-1 infection, while viral components were found in ESCRT+ EVs. Functional characterization of these EVs demonstrated that they have opposite effects on the infection, but the dominant effect was negative. Overall, we have identified the dominant population of EVs, and other EV populations produced during HSV-1 infection, and we have provided information about potential roles.IMPORTANCE Extracellular vesicles mediate cell-to-cell communication and convey messages important for cell homeostasis. Pathways of EV biogenesis are often hijacked by pathogens to facilitate their dissemination and to establish a favorable microenvironment for the infection. We have previously shown that HSV-1 infection alters the cargo and functions of the released EVs, which negatively impact the infection. We have built upon our previous findings by developing procedures to separate EV populations from HSV-1-infected cells. We identified the major population of EVs released during infection, which carries the DNA sensor STING and has an antiviral effect. We also identified an EV population that carries selected viral proteins and has a proviral role. This is the first study to characterize EV populations during infection. These data indicate that the complex interactions between the virus and the host are extended to the extracellular environment and could impact HSV-1 dissemination and persistence in the host.


Assuntos
Vesículas Extracelulares/fisiologia , Herpesvirus Humano 1/fisiologia , Antivirais/metabolismo , Linhagem Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Exocitose , Vesículas Extracelulares/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Proteínas de Membrana/metabolismo , Tetraspanina 30/metabolismo , Tetraspaninas/metabolismo , Proteínas Virais/metabolismo
7.
Elife ; 92020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33350386

RESUMO

Herpes simplex virus-1 (HSV-1) establishes a latent infection in neurons and periodically reactivates to cause disease. The stimuli that trigger HSV-1 reactivation have not been fully elucidated. We demonstrate HSV-1 reactivation from latently infected mouse neurons induced by forskolin requires neuronal excitation. Stimuli that directly induce neurons to become hyperexcitable also induced HSV-1 reactivation. Forskolin-induced reactivation was dependent on the neuronal pathway of DLK/JNK activation and included an initial wave of viral gene expression that was independent of histone demethylase activity and linked to histone phosphorylation. IL-1ß is released under conditions of stress, fever and UV exposure of the epidermis; all known triggers of clinical HSV reactivation. We found that IL-1ß induced histone phosphorylation and increased the excitation in sympathetic neurons. Importantly, IL-1ß triggered HSV-1 reactivation, which was dependent on DLK and neuronal excitability. Thus, HSV-1 co-opts an innate immune pathway resulting from IL-1 stimulation of neurons to induce reactivation.


Assuntos
Herpesvirus Humano 1/fisiologia , Interleucina-1beta/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Neurônios/virologia , Ativação Viral/fisiologia , Animais , Herpes Simples/imunologia , Herpes Simples/metabolismo , Camundongos , Latência Viral/fisiologia
8.
Viruses ; 13(1)2020 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-33374862

RESUMO

Viruses encode for structural proteins that participate in virion formation and include capsid and envelope proteins. In addition, viruses encode for an array of non-structural accessory proteins important for replication, spread, and immune evasion in the host and are often linked to virus pathogenesis. Most virus accessory proteins are non-essential for growth in cell culture because of the simplicity of the infection barriers or because they have roles only during a state of the infection that does not exist in cell cultures (i.e., tissue-specific functions), or finally because host factors in cell culture can complement their absence. For these reasons, the study of most nonessential viral factors is more complex and requires development of suitable cell culture systems and in vivo models. Approximately half of the proteins encoded by the herpes simplex virus 1 (HSV-1) genome have been classified as non-essential. These proteins have essential roles in vivo in counteracting antiviral responses, facilitating the spread of the virus from the sites of initial infection to the peripheral nervous system, where it establishes lifelong reservoirs, virus pathogenesis, and other regulatory roles during infection. Understanding the functions of the non-essential proteins of herpesviruses is important to understand mechanisms of viral pathogenesis but also to harness properties of these viruses for therapeutic purposes. Here, we have provided a comprehensive summary of the functions of HSV-1 non-essential proteins.


Assuntos
Regulação Viral da Expressão Gênica , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral , Animais , Endonucleases/genética , Endonucleases/metabolismo , Inativação Gênica , Herpes Simples/imunologia , Herpes Simples/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Ácidos Nucleicos/metabolismo , Fosfotransferases/genética , Fosfotransferases/metabolismo , Transativadores , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo , Virulência
9.
Cell Rep ; 33(5): 108339, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33147451

RESUMO

Here, we report our studies of immune-mediated regulation of Zika virus (ZIKV), herpes simplex virus 1 (HSV-1), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the human cornea. We find that ZIKV can be transmitted via corneal transplantation in mice. However, in human corneal explants, we report that ZIKV does not replicate efficiently and that SARS-CoV-2 does not replicate at all. Additionally, we demonstrate that type III interferon (IFN-λ) and its receptor (IFNλR1) are expressed in the corneal epithelium. Treatment of human corneal explants with IFN-λ, and treatment of mice with IFN-λ eye drops, upregulates antiviral interferon-stimulated genes. In human corneal explants, blockade of IFNλR1 enhances replication of ZIKV and HSV-1 but not SARS-CoV-2. In addition to an antiviral role for IFNλR1 in the cornea, our results suggest that the human cornea does not support SARS-CoV-2 infection despite expression of ACE2, a SARS-CoV-2 receptor, in the human corneal epithelium.


Assuntos
Betacoronavirus/fisiologia , Córnea/virologia , Infecções por Coronavirus/transmissão , Herpesvirus Humano 1/fisiologia , Interferons/imunologia , Pneumonia Viral/transmissão , Zika virus/fisiologia , Animais , Betacoronavirus/imunologia , Córnea/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Herpes Simples/imunologia , Herpes Simples/transmissão , Herpes Simples/virologia , Humanos , Camundongos , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Replicação Viral/fisiologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologia
10.
PLoS Pathog ; 16(8): e1008703, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776994

RESUMO

Herpes simplex virus type 1 (HSV1) is a complicated structural agent with a sophisticated transcription process and a high infection rate. A vaccine against HSV1 is urgently needed. As multiple viral-encoded proteins, including structural and nonstructural proteins, contribute to immune response stimulation, an attenuated or deficient HSV1 vaccine may be relatively reliable. Advances in genomic modification technologies provide reliable means of constructing various HSV vaccine candidates. Based on our previous work, an M6 mutant with mutations in the UL7, UL41, LAT, Us3, Us11 and Us12 genes was established. The mutant exhibited low proliferation in cells and an attenuated phenotype in an animal model. Furthermore, in mice and rhesus monkeys, the mutant can induce remarkable serum neutralizing antibody titers and T cell activation and protect against HSV1 challenge by impeding viral replication, dissemination and pathogenesis.


Assuntos
Vacinas contra o Vírus do Herpes Simples/imunologia , Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Animais , Feminino , Herpes Simples/prevenção & controle , Herpes Simples/virologia , Vacinas contra o Vírus do Herpes Simples/administração & dosagem , Vacinas contra o Vírus do Herpes Simples/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Fenótipo , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Proteínas Virais/administração & dosagem , Proteínas Virais/genética , Proteínas Virais/imunologia
11.
Anal Chem ; 92(16): 11297-11304, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32683857

RESUMO

Viruses are infections species that infect a large spectrum of living systems. Although displaying a wide variety of shapes and sizes, they are all composed of nucleic acid encapsulated into a protein capsid. After virions enter the host cell, they replicate to produce multiple copies of themselves. They then lyse the host, releasing virions to infect new cells. The high proliferation rate of viruses is the underlying cause of their fast transmission among living species. Although many viruses are harmless, some of them are responsible for severe diseases such as AIDS, viral hepatitis, and flu. Traditionally, electron microscopy is used to identify and characterize viruses. This approach is time- and labor-consuming, which is problematic upon pandemic proliferation of previously unknown viruses, such as H1N1 and COVID-19. Herein, we demonstrate a novel diagnosis approach for label-free identification and structural characterization of individual viruses that is based on a combination of nanoscale Raman and infrared spectroscopy. Using atomic force microscopy-infrared (AFM-IR) spectroscopy, we were able to probe structural organization of the virions of Herpes Simplex Type 1 viruses and bacteriophage MS2. We also showed that tip-enhanced Raman spectroscopy (TERS) could be used to reveal protein secondary structure and amino acid composition of the virus surface. Our results show that AFM-IR and TERS provide different but complementary information about the structure of complex biological specimens. This structural information can be used for fast and reliable identification of viruses. This nanoscale bimodal imaging approach can be also used to investigate the origin of viral polymorphism and study mechanisms of virion assembly.


Assuntos
Microscopia de Força Atômica/métodos , Nanoestruturas/química , Análise Espectral Raman/métodos , Vírion/química , Animais , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , Capsídeo/química , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Microscopia Crioeletrônica , Análise Discriminante , Herpesvirus Humano 1/fisiologia , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Análise dos Mínimos Quadrados , Levivirus/metabolismo , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Estrutura Terciária de Proteína , Células Vero
12.
Proc Natl Acad Sci U S A ; 117(32): 19475-19486, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32709741

RESUMO

The DNA sensor cGAS catalyzes the production of the cyclic dinucleotide cGAMP, resulting in type I interferon responses. We addressed the functionality of cGAS-mediated DNA sensing in human and murine T cells. Activated primary CD4+ T cells expressed cGAS and responded to plasmid DNA by upregulation of ISGs and release of bioactive interferon. In mouse T cells, cGAS KO ablated sensing of plasmid DNA, and TREX1 KO enabled cells to sense short immunostimulatory DNA. Expression of IFIT1 and MX2 was downregulated and upregulated in cGAS KO and TREX1 KO T cell lines, respectively, compared to parental cells. Despite their intact cGAS sensing pathway, human CD4+ T cells failed to mount a reverse transcriptase (RT) inhibitor-sensitive immune response following HIV-1 infection. In contrast, infection of human T cells with HSV-1 that is functionally deficient for the cGAS antagonist pUL41 (HSV-1ΔUL41N) resulted in a cGAS-dependent type I interferon response. In accordance with our results in primary CD4+ T cells, plasmid challenge or HSV-1ΔUL41N inoculation of T cell lines provoked an entirely cGAS-dependent type I interferon response, including IRF3 phosphorylation and expression of ISGs. In contrast, no RT-dependent interferon response was detected following transduction of T cell lines with VSV-G-pseudotyped lentiviral or gammaretroviral particles. Together, T cells are capable to raise a cGAS-dependent cell-intrinsic response to both plasmid DNA challenge or inoculation with HSV-1ΔUL41N. However, HIV-1 infection does not appear to trigger cGAS-mediated sensing of viral DNA in T cells, possibly by revealing viral DNA of insufficient quantity, length, and/or accessibility to cGAS.


Assuntos
Linfócitos T CD4-Positivos/virologia , HIV-1/fisiologia , Interferon Tipo I/metabolismo , Nucleotidiltransferases/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , DNA Viral/fisiologia , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/metabolismo , Camundongos , Nucleotidiltransferases/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Especificidade da Espécie , Replicação Viral
13.
J Virol ; 94(15)2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32461310

RESUMO

Herpes simplex virus 1 (HSV-1) establishes a lifelong latent infection in peripheral nerve ganglia. Periodically, the virus reactivates from this latent reservoir and is transported to the original site of infection. Strains of HSV-1 have been noted to vary greatly in their virulence and reactivation efficiencies in animal models. While HSV-1 strain 17syn + can be readily reactivated, strain KOS(M) shows little to no reactivation in the mouse and rabbit models of induced reactivation. Additionally, 17syn + is markedly more virulent in vivo than KOS. This has raised questions regarding potential strain-specific differences in neuroinvasion and neurovirulence and their contribution to differences in the establishment of latency (or ability to spread back to the periphery) and to the reactivation phenotype. To determine if any difference in the ability to reactivate between strains 17syn + and KOS(M) is manifest at the level of neurons, we utilized a recently characterized human neuronal cell line model of HSV latency and reactivation (LUHMES). We found that KOS(M) established latency with a higher number of viral genomes than strain 17syn + Strikingly, we show that the KOS(M) viral genomes have a higher burden of heterochromatin marks than strain 17syn + The increased heterochromatin profile for KOS(M) correlates with the reduced expression of viral lytic transcripts during latency and impaired induced reactivation compared to that of 17syn + These results suggest that genomes entering neurons from HSV-1 infections with strain KOS(M) are more prone to rapid heterochromatinization than those of 17syn + and that this results in a reduced ability to reactivate from latency.IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes a lifelong infection in neuronal cells. The virus periodically reactivates and causes recurrent disease. Strains of HSV-1 vary greatly in their virulence and potential to reactivate in animal models. Although these differences are phenotypically well defined, factors contributing to the strains' abilities to reactivate are largely unknown. We utilized a human neuronal cell line model of HSV latency and reactivation (LUHMES) to characterize the latent infection of two HSV-1 wild-type strains. We find that strain-specific differences in reactivation are recapitulated in LUHMES. Additionally, these differences correlate with the degree of heterochromatinization of the latent genomes. Our data suggest that the epigenetic state of the viral genome is an important determinant of reactivation that varies in a strain-specific manner. This work also shows the first evidence of strain-specific differences in reactivation outside the context of the whole animal at a human neuronal cell level.


Assuntos
Herpes Simples/metabolismo , Herpesvirus Humano 1/fisiologia , Modelos Biológicos , Neurônios/metabolismo , Ativação Viral/fisiologia , Latência Viral/fisiologia , Linhagem Celular , Herpes Simples/genética , Herpes Simples/patologia , Humanos , Neurônios/patologia , Neurônios/virologia
14.
Immunity ; 52(5): 767-781.e6, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32277911

RESUMO

The enzyme cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA in infected and malignant cells and catalyzes the formation of 2'3'cGMP-AMP (cGAMP), which in turn triggers interferon (IFN) production via the STING pathway. Here, we examined the contribution of anion channels to cGAMP transfer and anti-viral defense. A candidate screen revealed that inhibition of volume-regulated anion channels (VRACs) increased propagation of the DNA virus HSV-1 but not the RNA virus VSV. Chemical blockade or genetic ablation of LRRC8A/SWELL1, a VRAC subunit, resulted in defective IFN responses to HSV-1. Biochemical and electrophysiological analyses revealed that LRRC8A/LRRC8E-containing VRACs transport cGAMP and cyclic dinucleotides across the plasma membrane. Enhancing VRAC activity by hypotonic cell swelling, cisplatin, GTPγS, or the cytokines TNF or interleukin-1 increased STING-dependent IFN response to extracellular but not intracellular cGAMP. Lrrc8e-/- mice exhibited impaired IFN responses and compromised immunity to HSV-1. Our findings suggest that cell-to-cell transmission of cGAMP via LRRC8/VRAC channels is central to effective anti-viral immunity.


Assuntos
Fibroblastos/imunologia , Interferons/imunologia , Proteínas de Membrana/imunologia , Nucleotídeos Cíclicos/imunologia , Canais de Ânion Dependentes de Voltagem/imunologia , Animais , Antivirais/imunologia , Antivirais/metabolismo , Efeito Espectador , Linhagem Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HeLa , Herpes Simples/imunologia , Herpes Simples/virologia , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/fisiologia , Humanos , Interferons/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Nucleotidiltransferases/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo
15.
J Virol ; 94(15)2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32295910

RESUMO

During all stages of infection, herpes simplex virus 1 (HSV-1) expresses viral microRNAs (miRNAs). There are at least 20 confirmed HSV-1 miRNAs, yet the roles of individual miRNAs in the context of viral infection remain largely uncharacterized. We constructed a recombinant virus lacking the sequences for miR-H1-5p and miR-H6-3p (17dmiR-H1/H6). The seed sequences for these miRNAs are antisense to each other and are transcribed from divergent noncoding RNAs in the latency-associated transcript (LAT) promoter region. Comparing phenotypes exhibited by the recombinant virus lacking these miRNAs to the wild type (17syn+), we found that during acute infection in cell culture, 17dmiR-H1/H6 exhibited a modest increase in viral yields. Analysis of pathogenesis in the mouse following footpad infection revealed a slight increase in virulence for 17dmiR-H1/H6 but no significant difference in the establishment or maintenance of latency. Strikingly, explant of latently infected dorsal root ganglia revealed a decreased and delayed reactivation phenotype. Further, 17dmiR-H1/H6 was severely impaired in epinephrine-induced reactivation in the rabbit ocular model. Finally, we demonstrated that deletion of miR-H1/H6 increased the accumulation of the LAT as well as several of the LAT region miRNAs. These results suggest that miR-H1/H6 plays an important role in facilitating efficient reactivation from latency.IMPORTANCE While HSV antivirals reduce the severity and duration of clinical disease in some individuals, there is no vaccine or cure. Therefore, understanding the mechanisms regulating latency and reactivation as a potential to elucidate targets for better therapeutics is important. There are at least 20 confirmed HSV-1 miRNAs, yet the roles of individual miRNAs in the context of viral infection remain largely uncharacterized. The present study focuses on two of the miRNAs (miR-H1/H6) that are encoded within the latency-associated transcript (LAT) region, a portion of the genome that has been associated with efficient reactivation. Here, we demonstrate that the deletion of the seed sequences of these miRNAs results in a severe reduction in reactivation of HSV-1 in the mouse and rabbit models. These results suggest a linkage between these miRNAs and reactivation.


Assuntos
Gânglios Espinais/metabolismo , Herpes Simples/metabolismo , Herpesvirus Humano 1/fisiologia , MicroRNAs/metabolismo , RNA Viral/metabolismo , Ativação Viral , Latência Viral , Animais , Gânglios Espinais/virologia , Células HEK293 , Herpes Simples/genética , Humanos , Camundongos , MicroRNAs/genética , RNA Viral/genética , Coelhos
16.
PLoS One ; 15(3): e0230870, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32226020

RESUMO

When herpes simplex virus 1 (HSV-1) infection is initiated in the ocular, nasal, or oral cavity, sensory neurons within trigeminal ganglia (TG) become infected. Following a burst of viral transcription in TG neurons, lytic cycle viral genes are suppressed and latency is established. The latency-associated transcript (LAT) is the only viral gene abundantly expressed during latency, and LAT expression is important for the latency-reactivation cycle. Reactivation from latency is required for virus transmission and recurrent disease, including encephalitis. The Wnt/ß-catenin signaling pathway is differentially expressed in TG during the bovine herpesvirus 1 latency-reactivation cycle. Hence, we hypothesized HSV-1 regulates the Wnt/ß-catenin pathway and promotes maintenance of latency because this pathway enhances neuronal survival and axonal repair. New studies revealed ß-catenin was expressed in significantly more TG neurons during latency compared to TG from uninfected mice or mice latently infected with a LAT-/- mutant virus. When TG explants were incubated with media containing dexamethasone to stimulate reactivation, significantly fewer ß-catenin+ TG neurons were detected. Conversely, TG explants from uninfected mice or mice latently infected with a LAT-/- mutant increased the number of ß-catenin+ TG neurons in the presence of DEX relative to samples not treated with DEX. Impairing Wnt signaling with small molecule antagonists reduced virus shedding during explant-induced reactivation. These studies suggested ß-catenin was differentially expressed during the latency-reactivation cycle, in part due to LAT expression.


Assuntos
Regulação da Expressão Gênica , Herpesvirus Humano 1/fisiologia , Neurônios/metabolismo , Neurônios/virologia , Gânglio Trigeminal/citologia , Ativação Viral , beta Catenina/metabolismo , Animais , Feminino , Camundongos , Via de Sinalização Wnt
17.
Nat Commun ; 11(1): 1338, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165633

RESUMO

Macrophages have important protective functions during infection with herpes simplex virus type 1 (HSV-1). However, molecular mechanisms that restrict viral propagation and protect from severe disease are unclear. Here we show that macrophages take up HSV-1 via endocytosis and transport the virions into multivesicular bodies (MVBs). In MVBs, acid ceramidase (aCDase) converts ceramide into sphingosine and increases the formation of sphingosine-rich intraluminal vesicles (ILVs). Once HSV-1 particles reach MVBs, sphingosine-rich ILVs bind to HSV-1 particles, which restricts fusion with the limiting endosomal membrane and prevents cellular infection. Lack of aCDase in macrophage cultures or in Asah1-/- mice results in replication of HSV-1 and Asah1-/- mice die soon after systemic or intravaginal inoculation. The treatment of macrophages with sphingosine enhancing compounds blocks HSV-1 propagation, suggesting a therapeutic potential of this pathway. In conclusion, aCDase loads ILVs with sphingosine, which prevents HSV-1 capsids from penetrating into the cytosol.


Assuntos
Ceramidase Ácida/metabolismo , Herpes Simples/enzimologia , Herpes Simples/prevenção & controle , Herpesvirus Humano 1/fisiologia , Macrófagos/enzimologia , Corpos Multivesiculares/virologia , Ceramidase Ácida/genética , Animais , Feminino , Herpes Simples/virologia , Humanos , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Replicação Viral
18.
PLoS Pathog ; 16(3): e1008335, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32187211

RESUMO

One of the fundamental reactions of the innate immune responses to pathogen infection is the release of pro-inflammatory cytokines, including IL-1ß, processed by the NLRP3 inflammasome. The stimulator of interferon genes (STING) has the essential roles in innate immune response against pathogen infections. Here we reveal a distinct mechanism by which STING regulates the NLRP3 inflammasome activation, IL-1ß secretion, and inflammatory responses in human cell lines, mice primary cells, and mice. Interestingly, upon HSV-1 infection and cytosolic DNA stimulation, STING binds to NLRP3 and promotes the inflammasome activation through two approaches. First, STING recruits NLRP3 and facilitates NLRP3 localization in the endoplasmic reticulum, thereby facilitating the inflammasome formation. Second, STING interacts with NLRP3 and attenuates K48- and K63-linked polyubiquitination of NLRP3, thereby promoting the inflammasome activation. Collectively, we demonstrate that the cGAS-STING-NLRP3 signaling is essential for host defense against HSV-1 infection.


Assuntos
Retículo Endoplasmático/imunologia , Herpes Simples/imunologia , Inflamassomos/imunologia , Proteínas de Membrana/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Animais , Retículo Endoplasmático/metabolismo , Herpes Simples/genética , Herpes Simples/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Humanos , Imunidade Inata , Inflamassomos/genética , Inflamassomos/metabolismo , Macrófagos/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ligação Proteica , Transporte Proteico
19.
PLoS Pathog ; 16(3): e1008296, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32134994

RESUMO

A fundamental question in herpes simplex virus (HSV) pathogenesis is the consequence of viral reactivation to the neuron. Evidence supporting both post-reactivation survival and demise is published. The exceedingly rare nature of this event at the neuronal level in the sensory ganglion has limited direct examination of this important question. In this study, an in-depth in vivo analysis of the resolution of reactivation was undertaken. Latently infected C57BL/6 mice were induced to reactivate in vivo by hyperthermic stress. Infectious virus was detected in a high percentage (60-80%) of the trigeminal ganglia from these mice at 20 hours post-reactivation stimulus, but declined by 48 hours post-stimulus (0-13%). With increasing time post-reactivation stimulus, the percentage of reactivating neurons surrounded by a cellular cuff increased, which correlated with a decrease in detectable infectious virus and number of viral protein positive neurons. Importantly, in addition to intact viral protein positive neurons, fragmented viral protein positive neurons morphologically consistent with apoptotic bodies and containing cleaved caspase-3 were detected. The frequency of this phenotype increased through time post-reactivation. These fragmented neurons were surrounded by Iba1+ cells, consistent with phagocytic removal of dead neurons. Evidence of neuronal destruction post-reactivation prompted re-examination of the previously reported non-cytolytic role of T cells in controlling reactivation. Latently infected mice were treated with anti-CD4/CD8 antibodies prior to induced reactivation. Neither infectious virus titers nor neuronal fragmentation were altered. In contrast, when viral DNA replication was blocked during reactivation, fragmentation was not observed even though viral proteins were expressed. Our data demonstrate that at least a portion of reactivating neurons are destroyed. Although no evidence for direct T cell mediated antigen recognition in this process was apparent, inhibition of viral DNA replication blocked neuronal fragmentation. These unexpected findings raise new questions about the resolution of HSV reactivation in the host nervous system.


Assuntos
Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Neurônios/virologia , Ativação Viral , Animais , Apoptose , Caspase 3/genética , Caspase 3/metabolismo , Feminino , Herpes Simples/genética , Herpes Simples/metabolismo , Herpes Simples/fisiopatologia , Herpesvirus Humano 1/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Replicação Viral
20.
Sci Adv ; 6(12): eaaz3367, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32206724

RESUMO

Mammalian transient receptor potential (TRP) channels are major components of Ca2+ signaling pathways and control a diversity of physiological functions. Here, we report a specific role for TRPC1 in the entry of herpes simplex virus type 1 (HSV-1) into cells. HSV-1-induced Ca2+ release and entry were dependent on Orai1, STIM1, and TRPC1. Inhibition of Ca2+ entry or knockdown of these proteins attenuated viral entry and infection. HSV-1 glycoprotein D interacted with the third ectodomain of TRPC1, and this interaction facilitated viral entry. Knockout of TRPC1 attenuated HSV-1-induced ocular abnormality and morbidity in vivo in TRPC1-/- mice. There was a strong correlation between HSV-1 infection and plasma membrane localization of TRPC1 in epithelial cells within oral lesions in buccal biopsies from HSV-1-infected patients. Together, our findings demonstrate a critical role for TRPC1 in HSV-1 infection and suggest the channel as a potential target for anti-HSV therapy.


Assuntos
Herpes Simples/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Canais de Cátion TRPC/metabolismo , Internalização do Vírus , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Ativação do Canal Iônico , Camundongos , Modelos Biológicos , Mutação , Ligação Proteica , Canais de Cátion TRPC/genética , Células Vero
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