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1.
PLoS One ; 16(2): e0247396, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606823

RESUMO

Among various delivery systems for vaccine and drug delivery, cell-penetrating peptides (CPPs) have been known as a potent delivery system because of their capability to penetrate cell membranes and deliver some types of cargoes into cells. Several CPPs were found in the proteome of viruses such as Tat originated from human immunodeficiency virus-1 (HIV-1), and VP22 derived from herpes simplex virus-1 (HSV-1). In the current study, a wide-range of CPPs was identified in the proteome of SARS-CoV-2, a new member of coronaviruses family, using in silico analyses. These CPPs may play a main role for high penetration of virus into cells and infection of host. At first, we submitted the proteome of SARS-CoV-2 to CellPPD web server that resulted in a huge number of CPPs with ten residues in length. Afterward, we submitted the predicted CPPs to C2Pred web server for evaluation of the probability of each peptide. Then, the uptake efficiency of each peptide was investigated using CPPred-RF and MLCPP web servers. Next, the physicochemical properties of the predicted CPPs including net charge, theoretical isoelectric point (pI), amphipathicity, molecular weight, and water solubility were calculated using protparam and pepcalc tools. In addition, the probability of membrane binding potential and cellular localization of each CPP were estimated by Boman index using APD3 web server, D factor, and TMHMM web server. On the other hand, the immunogenicity, toxicity, allergenicity, hemolytic potency, and half-life of CPPs were predicted using various web servers. Finally, the tertiary structure and the helical wheel projection of some CPPs were predicted by PEP-FOLD3 and Heliquest web servers, respectively. These CPPs were divided into: a) CPP containing tumor homing motif (RGD) and/or tumor penetrating motif (RXXR); b) CPP with the highest Boman index; c) CPP with high half-life (~100 hour) in mammalian cells, and d) CPP with +5.00 net charge. Based on the results, we found a large number of novel CPPs with various features. Some of these CPPs possess tumor-specific motifs which can be evaluated in cancer therapy. Furthermore, the novel and potent CPPs derived from SARS-CoV-2 may be used alone or conjugated to some sequences such as nuclear localization sequence (NLS) for vaccine and drug delivery.


Assuntos
/química , Peptídeos Penetradores de Células/química , Biologia Computacional , Simulação por Computador , Sistemas de Liberação de Medicamentos , Proteoma , /química , Animais , /metabolismo , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/metabolismo , HIV-1/química , HIV-1/genética , Herpesvirus Humano 1/química , Herpesvirus Humano 1/genética , Humanos , /metabolismo , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
2.
PLoS Pathog ; 16(12): e1009166, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33370402

RESUMO

Herpes simplex virus 1 (HSV-1) infects skin and mucosal epithelial cells and then travels along axons to establish latency in the neurones of sensory ganglia. Although viral gene expression is restricted during latency, the latency-associated transcript (LAT) locus encodes many RNAs, including a 2 kb intron known as the hallmark of HSV-1 latency. Here, we studied HSV-1 infection and the role of the LAT locus in human skin xenografts in vivo and in cultured explants. We sequenced the genomes of our stock of HSV-1 strain 17syn+ and seven derived viruses and found nonsynonymous mutations in many viral proteins that had no impact on skin infection. In contrast, deletions in the LAT locus severely impaired HSV-1 replication and lesion formation in skin. However, skin replication was not affected by impaired intron splicing. Moreover, although the LAT locus has been implicated in regulating gene expression in neurones, we observed only small changes in transcript levels that were unrelated to the growth defect in skin, suggesting that its functions in skin may be different from those in neurones. Thus, although the LAT locus was previously thought to be dispensable for lytic infection, we show that it is a determinant of HSV-1 virulence during lytic infection of human skin.


Assuntos
Herpes Simples/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/patogenicidade , MicroRNAs/genética , Pele/virologia , Virulência/genética , Animais , Xenoenxertos , Humanos , Camundongos , Fatores de Virulência/genética
3.
Viruses ; 12(12)2020 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-33322225

RESUMO

Acyclovir is the drug of choice for the treatment of herpes simplex virus (HSV) infections. Acyclovir-resistant HSV strains may emerge, especially during long-term drug use, and subsequently cause difficult-to-treat exacerbations. Previously, we set up a novel treatment approach, based on enzymatically synthesized pools of siRNAs, or siRNA swarms. These swarms can cover kilobases-long target sequences, reducing the likelihood of resistance to treatment. Swarms targeting the UL29 essential gene of HSV-1 have demonstrated high efficacy against HSV-1 in vitro and in vivo. Here, we assessed the antiviral potential of a UL29 siRNA swarm against circulating strains of HSV-1, in comparison with acyclovir. All circulating strains were sensitive to both antivirals, with the half-maximal inhibitory concentrations (IC50) in the range of 350-1911 nM for acyclovir and 0.5-3 nM for the UL29 siRNA swarm. Additionally, we showed that an acyclovir-resistant HSV-1, devoid of thymidine kinase, is highly sensitive to UL29 siRNA treatment (IC50 1.0 nM; Imax 97%). Moreover, the detected minor variations in the RNAi target of the HSV strains had no effect on the potency or efficacy of UL29 siRNA swarm treatment. Our findings support the development of siRNA swarms for the treatment of HSV-1 infections, in order to circumvent any potential acyclovir resistance.


Assuntos
Aciclovir/farmacologia , Proteínas de Ligação a DNA/genética , Herpes Simples/virologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Virais/genética , Aciclovir/uso terapêutico , Animais , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Herpes Simples/terapia , Herpesvirus Humano 1/classificação , Herpesvirus Humano 1/isolamento & purificação , Humanos , Concentração Inibidora 50 , Células Vero
4.
Nat Commun ; 11(1): 4894, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994400

RESUMO

Identification of the complete set of translated genes of viruses is important to understand viral replication and pathogenesis as well as for therapeutic approaches to control viral infection. Here, we use chemical proteomics, integrating bio-orthogonal non-canonical amino acid tagging and high-resolution mass spectrometry, to characterize the newly synthesized herpes simplex virus 1 (HSV-1) proteome in infected cells. In these infected cells, host cellular protein synthesis is shut-off, increasing the chance to preferentially detect viral proteomes. We identify nine previously cryptic orphan protein coding sequences whose translated products are expressed in HSV-1-infected cells. Functional characterization of one identified protein, designated piUL49, shows that it is critical for HSV-1 neurovirulence in vivo by regulating the activity of virally encoded dUTPase, a key enzyme that maintains accurate DNA replication. Our results demonstrate that cryptic orphan protein coding genes of HSV-1, and probably other large DNA viruses, remain to be identified.


Assuntos
Encefalite por Herpes Simples/virologia , Herpesvirus Humano 1/patogenicidade , Pirofosfatases/metabolismo , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Animais , Encéfalo/patologia , Encéfalo/virologia , Chlorocebus aethiops , Replicação do DNA , Modelos Animais de Doenças , Encefalite por Herpes Simples/patologia , Feminino , Genes Virais/genética , Células HEK293 , Células HeLa , Herpesvirus Humano 1/genética , Humanos , Camundongos , Biossíntese de Proteínas , Proteômica/métodos , Células Vero , Proteínas Virais/genética , Fatores de Virulência/genética , Replicação Viral
5.
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
6.
Nat Commun ; 11(1): 4148, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811834

RESUMO

We evaluate gene editing of HSV in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection. Here we show that AAV-delivered meganucleases, but not CRISPR/Cas9, mediate highly efficient gene editing of HSV, eliminating over 90% of latent virus from superior cervical ganglia. Single-cell RNA sequencing demonstrates that both HSV and individual AAV serotypes are non-randomly distributed among neuronal subsets in ganglia, implying that improved delivery to all neuronal subsets may lead to even more complete elimination of HSV. As predicted, delivery of meganucleases using a triple AAV serotype combination results in the greatest decrease in ganglionic HSV loads. The levels of HSV elimination observed in these studies, if translated to humans, would likely significantly reduce HSV reactivation, shedding, and lesions. Further optimization of meganuclease delivery and activity is likely possible, and may offer a pathway to a cure for HSV infection.


Assuntos
Desoxirribonucleases/genética , Dependovirus/genética , Infecções Oculares/terapia , Edição de Genes/métodos , Herpes Simples/terapia , Herpesvirus Humano 1/genética , Latência Viral/genética , Animais , Sistemas CRISPR-Cas/genética , Células Cultivadas , Chlorocebus aethiops , Infecções Oculares/genética , Infecções Oculares/virologia , Feminino , Células HEK293 , Herpes Simples/genética , Herpesvirus Humano 1/patogenicidade , Humanos , Camundongos , Neurônios/metabolismo , Neurônios/virologia , RNA-Seq , Análise de Célula Única , Gânglio Cervical Superior/metabolismo , Gânglio Cervical Superior/virologia , Células Vero
7.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32611749

RESUMO

Us3 proteins of herpes simplex virus 1 (HSV-1) and HSV-2 are multifunctional serine-threonine protein kinases. Here, we identified an HSV-2 tegument protein, UL7, as a novel physiological substrate of HSV-2 Us3. Mutations in HSV-2 UL7, which precluded Us3 phosphorylation of the viral protein, significantly reduced mortality, viral replication in the vagina, and development of vaginal disease in mice following vaginal infection. These results indicated that Us3 phosphorylation of UL7 in HSV-2 was required for efficient viral replication and pathogenicity in vivo Of note, this phosphorylation was conserved in UL7 of chimpanzee herpesvirus (ChHV), which phylogenetically forms a monophyletic group with HSV-2 and the resurrected last common ancestral UL7 for HSV-2 and ChHV. In contrast, the phosphorylation was not conserved in UL7s of HSV-1, which belongs to a sister clade of the monophyletic group, the resurrected last common ancestor for HSV-1, HSV-2, and ChHV, and other members of the genus Simplexvirus that are phylogenetically close to these viruses. Thus, evolution of Us3 phosphorylation of UL7 coincided with the phylogeny of simplex viruses. Furthermore, artificially induced Us3 phosphorylation of UL7 in HSV-1, in contrast to phosphorylation in HSV-2, had no effect on viral replication and pathogenicity in mice. Our results suggest that HSV-2 and ChHV have acquired and maintained Us3 phosphoregulation of UL7 during their evolution because the phosphoregulation had an impact on viral fitness in vivo, whereas most other simplex viruses have not because the phosphorylation was not necessary for efficient fitness of the viruses in vivo IMPORTANCE It has been hypothesized that the evolution of protein phosphoregulation drives phenotypic diversity across species of organisms, which impacts fitness during their evolution. However, there is a lack of information regarding linkage between the evolution of viral phosphoregulation and the phylogeny of virus species. In this study, we clarified the novel HSV-2 Us3 phosphoregulation of UL7 in infected cells, which is important for viral replication and pathogenicity in vivo We also showed that the evolution of Us3 phosphoregulation of UL7 was linked to the phylogeny of viruses that are phylogenetically close to HSV-2 and to the phosphorylation requirements for the efficient in vivo viral fitness of HSV-2 and HSV-1, which are representative of viruses that have and have not evolved phosphoregulation, respectively. This study reports the first evidence showing that evolution of viral phosphoregulation coincides with phylogeny of virus species and supports the hypothesis regarding the evolution of viral phosphoregulation during viral evolution.


Assuntos
Regulação Viral da Expressão Gênica , Herpes Genital/virologia , Herpesvirus Humano 2/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas da Matriz Viral/genética , Proteínas Virais/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Modelos Animais de Doenças , Evolução Molecular , Feminino , Aptidão Genética , Células HEK293 , Herpes Genital/mortalidade , Herpesvirus Humano 1/classificação , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/patogenicidade , Herpesvirus Humano 2/classificação , Herpesvirus Humano 2/metabolismo , Herpesvirus Humano 2/patogenicidade , Humanos , Camundongos , Fosforilação , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Vagina/virologia , Células Vero , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , Virulência , Replicação Viral
8.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32669329

RESUMO

Herpesviruses exist in nature within each host animal. Ten herpesviruses have been isolated from bats and their biological properties reported. A novel bat alphaherpesvirus, which we propose to name "Pteropus lylei-associated alphaherpesvirus (PLAHV)," was isolated from urine of the fruit bat Pteropus lylei in Vietnam and characterized. The entire genome sequence was determined to be 144,008 bp in length and predicted to include 72 genes. PLAHV was assigned to genus Simplexvirus with other bat alphaherpesviruses isolated from pteropodid bats in Southeast Asia and Africa. The replication capacity of PLAHV in several cells was evaluated in comparison with that of herpes simplex virus 1 (HSV-1). PLAHV replicated better in the bat-originated cell line and less in human embryonic lung fibroblasts than HSV-1 did. PLAHV was serologically related to another bat alphaherpesvirus, Pteropodid alphaherpesvirus 1 (PtAHV1), isolated from a Pteropus hypomelanus-related bat captured in Indonesia, but not with HSV-1. PLAHV caused lethal infection in mice. PLAHV was as susceptible to acyclovir as HSV-1 was. Characterization of this new member of bat alphaherpesviruses, PLAHV, expands the knowledge on bat-associated alphaherpesvirology.IMPORTANCE A novel bat alphaherpesvirus, Pteropus lylei-associated alphaherpesvirus (PLAHV), was isolated from urine of the fruit bat Pteropus lylei in Vietnam. The whole-genome sequence was determined and was predicted to include 72 open reading frames in the 144,008-bp genome. PLAHV is circulating in a species of fruit bats, Pteropus lylei, in Asia. This study expands the knowledge on bat-associated alphaherpesvirology.


Assuntos
Alphaherpesvirinae/genética , Quirópteros/virologia , Genoma Viral , Infecções por Herpesviridae/veterinária , Proteínas Virais/genética , Aciclovir/farmacologia , Alphaherpesvirinae/classificação , Alphaherpesvirinae/efeitos dos fármacos , Alphaherpesvirinae/patogenicidade , Animais , Antivirais/farmacologia , Células COS , Linhagem Celular , Chlorocebus aethiops , Fibroblastos/virologia , Expressão Gênica , Tamanho do Genoma , Células HeLa , Infecções por Herpesviridae/tratamento farmacológico , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/mortalidade , Herpesvirus Humano 1/classificação , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/crescimento & desenvolvimento , Herpesvirus Humano 1/patogenicidade , Humanos , Camundongos , Filogenia , Análise de Sobrevida , Células Vero , Vietnã/epidemiologia , Proteínas Virais/metabolismo , Replicação Viral
10.
Nat Immunol ; 21(7): 727-735, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32541831

RESUMO

Stimulator-of-interferon genes (STING) is vital for sensing cytosolic DNA and initiating innate immune responses against microbial infection and tumors. Redox homeostasis is the balance of oxidative and reducing reactions present in all living systems. Yet, how the intracellular redox state controls STING activation is unclear. Here, we show that cellular redox homeostasis maintained by glutathione peroxidase 4 (GPX4) is required for STING activation. GPX4 deficiency enhanced cellular lipid peroxidation and thus specifically inhibited the cGAS-STING pathway. Concordantly, GPX4 deficiency inhibited herpes simplex virus-1 (HSV-1)-induced innate antiviral immune responses and promoted HSV-1 replication in vivo. Mechanistically, GPX4 inactivation increased production of lipid peroxidation, which led to STING carbonylation at C88 and inhibited its trafficking from the endoplasmic reticulum (ER) to the Golgi complex. Thus, cellular stress-induced lipid peroxidation specifically attenuates the STING DNA-sensing pathway, suggesting that GPX4 facilitates STING activation by maintaining redox homeostasis of lipids.


Assuntos
Herpes Simples/imunologia , Proteínas de Membrana/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Animais , Carbolinas/farmacologia , Células Cultivadas , DNA Viral/imunologia , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Feminino , Fibroblastos , Complexo de Golgi/metabolismo , Células HEK293 , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Homeostase/imunologia , Humanos , Imunidade Inata , Peroxidação de Lipídeos/genética , Peroxidação de Lipídeos/imunologia , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Nucleotidiltransferases/metabolismo , Oxirredução , Oximas/farmacologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/antagonistas & inibidores , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Cultura Primária de Células , Carbonilação Proteica/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Sulfonamidas/farmacologia , Células THP-1 , Replicação Viral/imunologia
11.
J Virol ; 94(14)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376626

RESUMO

Expression of viral genes and activation of innate antiviral responses during infection result in an increase in reactive oxygen species (ROS) and toxic by-products of energy metabolism which can lead to cell death. The mitochondrion and its associated proteins are crucial regulators of these responses and related pathways such as autophagy and apoptosis. Through a mass spectrometry approach, we have shown that the herpes simplex virus 1 (HSV-1) neurovirulence- and autophagy-modulating protein ICP34.5 interacts with numerous mitochondrion-associated factors. Specifically, we showed that amino acids 68 to 87 of ICP34.5, the domain that binds beclin1 and controls neurovirulence, are necessary for interactions with PGAM5, KEAP1, and other regulators of the antioxidant response, mitochondrial trafficking, and programmed cell death. We further show that while this domain interacts with multiple cellular stress response factors, it does not alter apoptosis or antioxidant gene expression. That said, the attenuated replication of a recombinant virus lacking residues 68 to 87 (termed Δ68-87) in primary human fibroblasts was restored by addition of ferric nitrate. Furthermore, in primary mouse neurons, the perinuclear localization of mitochondria that follows infection with HSV-1 was notably absent following Δ68-87 infection. Through this 20-amino-acid domain, ICP34.5 significantly reduces mitochondrial motility in axons of neurons. We propose the hypothesis that ICP34.5 promotes perinuclear mitochondrial localization by modulating transport of mitochondria through interaction with PGAM5. These data expand upon previous observations of altered mitochondrial dynamics following alphaherpesvirus infections and identify a key determinant of this activity during HSV-1 infections.IMPORTANCE Herpes simplex virus persists lifelong in neurons and can reactivate to cause recurrent lesions in mucosal tissues. A key determinant of virulence is the viral protein ICP34.5, of which residues 68 to 87 significantly contribute to neurovirulence through an unknown mechanism. Our report provides evidence that residues 68 to 87 of ICP34.5 are required for binding mitochondrion-associated factors. These interactions alter mitochondrial dynamics in neurons, thereby facilitating viral replication and pathogenesis.


Assuntos
Axônios/metabolismo , Herpes Simples/metabolismo , Herpesvirus Humano 1/metabolismo , Mitocôndrias/metabolismo , Proteínas Virais/metabolismo , Axônios/patologia , Axônios/virologia , Células HEK293 , Herpes Simples/patologia , Herpesvirus Humano 1/genética , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Domínios Proteicos , Transporte Proteico , Proteínas Virais/genética
12.
Nat Commun ; 11(1): 2038, 2020 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-32341360

RESUMO

The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution.


Assuntos
Genoma Viral , Herpesvirus Humano 1/genética , Animais , Produtos Biológicos/farmacologia , Chlorocebus aethiops , Biologia Computacional , Cricetinae , Fibroblastos/metabolismo , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Genes Virais , Genômica , Herpesvirus Humano 1/efeitos dos fármacos , Humanos , Fases de Leitura Aberta , Domínios Proteicos , Isoformas de Proteínas , Ribossomos/metabolismo , Transcriptoma , Células Vero
13.
J Neuroimmunol ; 344: 577241, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32334204

RESUMO

BACKGROUND: The cerebral innate immune system has a critical role in control processes of viral replication in the brain after primary infactivo and immunologic disregulation and inflammation has been reported as a primary determinant of pathogenesis and prognosis of subsequent HSV-1 related encephalitis (HSE). Interaction linking LTR3-activated DCs is also represented by the killer Ig-like receptor (KIR) + pathways on NK cells. Only a few studies analyzed the role of of MMP-9 activity regulating genetic polymorphism on clinical outcome of viral infections. Susceptibility to symptomatic encephalitis depends on SNC viral invasion and BBB disruption. We hypothesize that certain KIR genes and MMP allele may help to characterize a risk profile of developing an acute encephalitis due to HSV 1. AIM OF THE STUDY: Analyze the frequency of KIR genes and the C(-1562)T MMP-9 allels in subjects with HSV-1 encephalitis and to analyze their interaction with regard of the risk of occurrence of a symptomatic encephalitis. MATERIALS AND METHODS: Between November 2014 and January 2019, all consecutive patients with symptomatic acute encephalitis were recruited from three wards (Internal Medicine, Neurology, and Infectious Diseases) of "P. Giaccone" University Hospital, Palermo. RESULTS: Patients with acute viral encephalitis in comparison to controls showed a higher frequency AA KIR haplotype, HLA-C2 and of HLA-A-Bw4 alleles. With regard of HLA allele frequency patients with acute viral encephalitis In comparison to controls also showed a higher frequency of HLA-C2 and of HLA-A-Bw4 alleles. With regard of MMP-9 alleles, subjects with acute viral encephalitis were more likely to have the TT genotype. The multiple logistic regression analysis considering variables predictive of the occurrence of acute viral encephalitis showed the detrimental effect of AA KIR, HLAC1, HLA-A-BW4 and HLA-B-BW4T and of TT aplotype of MMP-9 genotype. CONCLUSIONS: Our study shows that in immunocompetent adult subjects there is an association between some KIR genes, MMP-9 alleles and HLA-ligand alleles and susceptibility to develop a symptomatic acute viral encephalitis. Definition of the genetic and immunological background of acute viral encephalitis can play a key role to determine personalized medicine.


Assuntos
Encefalite Viral/genética , Antígenos HLA/genética , Herpes Simples/genética , Herpesvirus Humano 1/genética , Metaloproteinase 9 da Matriz/genética , Receptores KIR/genética , Idoso , Estudos de Coortes , Encefalite Viral/diagnóstico , Encefalite Viral/metabolismo , Feminino , Antígenos HLA/metabolismo , Herpes Simples/diagnóstico , Herpes Simples/metabolismo , Humanos , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteases/genética , Metaloproteases/metabolismo , Pessoa de Meia-Idade , Receptores KIR/metabolismo
14.
J Virol ; 94(12)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32295906

RESUMO

ND10 nuclear bodies, as part of the intrinsic defenses, impose repression on incoming DNA. Infected cell protein 0 (ICP0), an E3 ubiquitin ligase of herpes simplex virus 1 (HSV-1), can derepress viral genes by degrading ND10 organizers to disrupt ND10. These events are part of the initial tug of war between HSV-1 and host, which determines the ultimate outcome of infection. Previously, we reported that ICP0 differentially recognizes promyelocytic leukemia (PML) isoforms. ICP0 depends on a SUMO-interaction motif located at residues 362 to 364 (SIM362-364) to trigger the degradation of PML isoforms II, IV, and VI, while using a bipartite sequence flanking the RING domain to degrade PML I. In this study, we investigated how the SUMO-SIM interaction regulates the degradation of PML II and PML II-associated proteins in ND10. We found that (i) the same regulatory mechanism for PML II degradation was detected in cells permissive or nonpermissive to the ICP0-null virus; (ii) the loss of a single SIM362-364 motif was restored by the presence of four consecutive SIMs from RNF4, but was not rescued by only two of the RNF4 SIMs; (iii) the loss of three C-terminal SIMs of ICP0 was fully restored by four RNF4 SIMs and also partially rescued by two RNF4 SIMs; and (iv) a PML II mutant lacking both lysine SUMOylation and SIM was not recognized by ICP0 for degradation, but was localized to ND10 and mitigated the degradation of other ND10 components, leading to delayed viral production. Taken together, SUMO regulates ICP0 substrate recognition via multiple fine-tuned mechanisms in HSV-1 infection.IMPORTANCE HSV-1 ICP0 is a multifunctional immediate early protein key to effective replication in the HSV-1 lytic cycle and reactivation in the latent cycle. ICP0 transactivates gene expression by orchestrating an overall mitigation in host intrinsic/innate restrictions. How ICP0 coordinates its multiple active domains and its diverse protein-protein interactions is a key question in understanding the HSV-1 life cycle and pathogenesis. The present study focuses on delineating the regulatory effects of the SUMO-SIM interaction on ICP0 E3 ubiquitin ligase activity regarding PML II degradation. For the first time, we discovered the importance of multivalency in the PML II-ICP0 interaction network and report the involvement of different regulatory mechanisms in PML II recognition by ICP0 in HSV-1 infection.


Assuntos
Herpesvirus Humano 1/imunologia , Interações Hospedeiro-Patógeno/imunologia , Proteínas Imediatamente Precoces/imunologia , Proteínas Nucleares/imunologia , Proteína da Leucemia Promielocítica/imunologia , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/imunologia , Ubiquitina-Proteína Ligases/imunologia , Linhagem Celular Tumoral , Células Epiteliais/imunologia , Células Epiteliais/virologia , Regulação da Expressão Gênica , Herpesvirus Humano 1/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas Imediatamente Precoces/genética , Mutação , Proteínas Nucleares/genética , Proteína da Leucemia Promielocítica/genética , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Proteólise , Transdução de Sinais , Sumoilação , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética
15.
PLoS Pathog ; 16(3): e1008445, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32226043

RESUMO

Herpesviral encephalitis caused by Herpes Simplex Virus 1 (HSV-1) is one of the most devastating diseases in humans. Patients present with fever, mental status changes or seizures and when untreated, sequelae can be fatal. Herpes Simplex Encephalitis (HSE) is characterized by mainly unilateral necrotizing inflammation effacing the frontal and mesiotemporal lobes with rare involvement of the brainstem. HSV-1 is hypothesized to invade the CNS via the trigeminal or olfactory nerve, but viral tropism and the exact route of infection remain unclear. Several mouse models for HSE have been developed, but they mimic natural infection only inadequately. The porcine alphaherpesvirus Pseudorabies virus (PrV) is closely related to HSV-1 and Varicella Zoster Virus (VZV). While pigs can control productive infection, it is lethal in other susceptible animals associated with severe pruritus leading to automutilation. Here, we describe the first mutant PrV establishing productive infection in mice that the animals are able to control. After intranasal inoculation with a PrV mutant lacking tegument protein pUL21 and pUS3 kinase activity (PrV-ΔUL21/US3Δkin), nearly all mice survived despite extensive infection of the central nervous system. Neuroinvasion mainly occurred along the trigeminal pathway. Whereas trigeminal first and second order neurons and autonomic ganglia were positive early after intranasal infection, PrV-specific antigen was mainly detectable in the frontal, mesiotemporal and parietal lobes at later times, accompanied by a long lasting lymphohistiocytic meningoencephalitis. Despite this extensive infection, mice showed only mild to moderate clinical signs, developed alopecic skin lesions, or remained asymptomatic. Interestingly, most mice exhibited abnormalities in behavior and activity levels including slow movements, akinesia and stargazing. In summary, clinical signs, distribution of viral antigen and inflammatory pattern show striking analogies to human encephalitis caused by HSV-1 or VZV not observed in other animal models of disease.


Assuntos
Encefalite por Varicela Zoster , Gânglios Autônomos , Herpes Simples , Herpesvirus Humano 1 , Herpesvirus Suídeo 1 , Herpesvirus Humano 3 , Neurônios , Pseudorraiva , Animais , Modelos Animais de Doenças , Encefalite por Varicela Zoster/genética , Encefalite por Varicela Zoster/metabolismo , Feminino , Gânglios Autônomos/metabolismo , Gânglios Autônomos/patologia , Gânglios Autônomos/virologia , Herpes Simples/genética , Herpes Simples/metabolismo , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/metabolismo , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/metabolismo , Humanos , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Neurônios/virologia , Pseudorraiva/genética , Pseudorraiva/metabolismo , Pseudorraiva/patologia , Suínos
16.
Sci Rep ; 10(1): 4307, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152425

RESUMO

Oncolytic virotherapy is emerging as a promising therapeutic option for solid tumours. Several oncolytic vectors in clinical testing are based on attenuated viruses; thus, efforts are being taken to develop a new repertoire of oncolytic viruses, based on virulent viral genomes. This possibility, however, raises concerns dealing with the safety features of the virulent phenotypes. We generated a double regulated Herpes simplex type-1 virus (HSV-1), in which tumour cell restricted replicative potential was combined to selective entry via ERBB2 receptor retargeting. The transcriptional control of the viral alpha4 gene encoding for the infected cell protein-4 (ICP4) by the cellular Survivin/BIRC5 promoter conferred a tumour cell-restricted replicative potential to a virulent HSV-1 genome. The combination of the additional ERBB2 retargeting further improved the selectivity for tumour cells, conferring to the double regulated virus a very limited ability to infect and propagate in non-cancerous cells. Accordingly, a suitable replicative and cytotoxic potential was maintained in tumour cell lines, allowing the double regulated virus to synergize in vivo with immune checkpoint (anti-PD-1) blockade in immunocompetent mice. Thus, restricting the replicative spectrum and tropism of virulent HSV-1 genomes by combination of conditional replication and retargeting provides an improved safety, does not alter the oncolytic strength, and is exploitable for its therapeutic potential with immune checkpoint blockade in cancer.


Assuntos
Herpesvirus Humano 1/genética , Terapia Viral Oncolítica/métodos , Neoplasias Ovarianas/terapia , Regiões Promotoras Genéticas , Receptor ErbB-2/metabolismo , Survivina/genética , Replicação Viral , Animais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/virologia , Receptor ErbB-2/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
17.
PLoS Pathog ; 16(3): e1008387, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32126128

RESUMO

Mediator of IRF3 activation (MITA, also named as STING/ERIS/MPYS/TMEM173), is essential to DNA virus- or cytosolic DNA-triggered innate immune responses. In this study, we demonstrated the negative regulatory role of RING-finger protein (RNF) 90 in innate immune responses targeting MITA. RNF90 promoted K48-linked ubiquitination of MITA and its proteasome-dependent degradation. Overexpression of RNF90 inhibited HSV-1- or cytosolic DNA-induced immune responses whereas RNF90 knockdown had the opposite effects. Moreover, RNF90-deficient bone marrow-derived dendritic cells (BMDCs), bone marrow-derived macrophages (BMMs) and mouse embryonic fibroblasts (MEFs) exhibited increased DNA virus- or cytosolic DNA-triggered signaling and RNF90 deficiency protected mice from DNA virus infection. Taken together, our findings suggested a novel function of RNF90 in innate immunity.


Assuntos
Herpesvirus Humano 1/imunologia , Imunidade Inata , Proteínas de Membrana/imunologia , Proteólise , Proteínas com Motivo Tripartido/imunologia , Ubiquitina-Proteína Ligases/imunologia , Ubiquitinação/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/virologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Fibroblastos/imunologia , Fibroblastos/virologia , Herpesvirus Humano 1/genética , Macrófagos/imunologia , Macrófagos/virologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética
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.
BMC Infect Dis ; 20(1): 150, 2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-32070282

RESUMO

BACKGROUND: Human encephalitis can originate from a variety of different aetiologies, of which infection is the most common one. The diagnostic work-up is specifically challenging in patients with travel history since a broader spectrum of unfamiliar additional infectious agents, e. g. tropical disease pathogens, needs to be considered. Here we present a case of encephalitis of unclear aetiology in a female traveller returning from Africa, who in addition developed an atypical herpes simplex virus (HSV) encephalitis in close temporal relation with high-dose steroid treatment. CASE PRESENTATION: A previously healthy 48-year-old female presented with confusion syndrome and impaired vigilance which had developed during a six-day trip to The Gambia. The condition rapidly worsened to a comatose state. Extensive search for infectious agents including a variety of tropical disease pathogens was unsuccessful. As encephalitic signs persisted despite of calculated antimicrobial and antiviral therapy, high-dose corticosteroids were applied intravenously based on the working diagnosis of an autoimmune encephalitis. The treatment did, however, not improve the patient's condition. Four days later, bihemispheric signal amplification in the insular and frontobasal cortex was observed on magnetic resonance imaging (MRI). The intracranial pressure rapidly increased and could not be controlled by conservative treatment. The patient died due to tonsillar herniation 21 days after onset of symptoms. Histological examination of postmortem brain tissue demonstrated a generalized lymphocytic meningoencephalitis. Immunohistochemical reactions against HSV-1/2 indicated an atypical manifestation of herpesviral encephalitis in brain tissue. Moreover, HSV-1 DNA was detected by a next-generation sequencing (NGS) metagenomics approach. Retrospective analysis of cerebrospinal fluid (CSF) and serum samples revealed HSV-1 DNA only in specimens one day ante mortem. CONCLUSIONS: This case shows that standard high-dose steroid therapy can contribute to or possibly even trigger fulminant cerebral HSV reactivation in a critically ill patient. Thus, even if extensive laboratory diagnostics including wide-ranging search for infectious pathogens has been performed before and remained without results, continuous re-evaluation of potential differential diagnoses especially regarding opportunistic infections or reactivation of latent infections is of utmost importance, particularly if new symptoms occur.


Assuntos
Encefalite por Herpes Simples/diagnóstico , Encefalite por Herpes Simples/tratamento farmacológico , Encefalite por Herpes Simples/etiologia , Herpes Simples/diagnóstico , Herpesvirus Humano 1/isolamento & purificação , Esteroides/efeitos adversos , Autopsia , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , DNA Viral/sangue , DNA Viral/líquido cefalorraquidiano , Encefalite/diagnóstico , Feminino , Gâmbia , Doença de Hashimoto/diagnóstico , Herpes Simples/diagnóstico por imagem , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/patogenicidade , Herpesvirus Humano 2/patogenicidade , Humanos , Imagem por Ressonância Magnética , Pessoa de Meia-Idade , Estudos Retrospectivos , Esteroides/uso terapêutico , Viagem
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