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Moloney leukemia virus 10 protein (MOV10) is an interferon (IFN)-inducible RNA helicase implicated in antiviral activity against RNA viruses, yet its role in herpesvirus infection has not been investigated. After corneal inoculation of mice with herpes simplex virus 1 (HSV-1), we observed strong upregulation of both MOV10 mRNA and protein in acutely infected mouse trigeminal ganglia. MOV10 suppressed HSV-1 replication in both neuronal and non-neuronal cells, and this suppression required the N-terminus, but not C-terminal helicase domain of MOV10. MOV10 repressed expression of the viral gene ICP0 in transfected cells, but suppressed HSV-1 replication independently of ICP0. MOV10 increased expression of type I IFN in HSV-1 infected cells with little effect on IFN downstream signaling. Treating the cells with IFN-α or an inhibitor of the IFN receptor eliminated MOV10 suppression of HSV-1 replication. MOV10 enhanced IFN production stimulated by cytoplasmic RNA rather than DNA. IKKε co-immunoprecipitated with MOV10 and was required for MOV10 restriction of HSV-1 replication. Mass spectrometry identified ICP27 as a viral protein interacting with MOV10. Co-immunoprecipitation results suggested that this interaction depended on the RGG box of ICP27 and both termini of MOV10. Overexpressed ICP27, but not its RGG-Box deletion mutant, rendered MOV10 unable to regulate HSV-1 replication and type I IFN production. In summary, MOV10 is induced to restrict HSV-1 lytic infection by promoting the type I IFN response through an IKKε-mediated RNA sensing pathway, and its activity is potentially antagonized by ICP27 in an RGG box dependent manner.
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Herpes Simples , Herpesvirus Humano 1 , Proteínas Imediatamente Precoces , Interferon Tipo I , Animais , Herpes Simples/genética , Herpesvirus Humano 1/fisiologia , Quinase I-kappa B , Proteínas Imediatamente Precoces/metabolismo , Camundongos , RNA , Replicação ViralRESUMO
Real-time imaging tools for single-virus tracking provide spatially resolved, quantitative measurements of viral replication and virus-host interactions. However, efficiently labeling both parental and progeny viruses in living host cells remains challenging. Here, we developed a novel strategy using the CRISPR-Tag system to detect herpes simplex virus 1 (HSV-1) DNA in host cells. We created recombinant HSV-1 harboring an ~600-bp CRISPR-Tag sequence which can be sufficiently recognized by dCas9-fluorescent protein (FP) fusion proteins. CRISPR-assisted single viral genome tracking (CASVIT) allows us to assess the temporal and spatial information of viral replication at the single-cell level. Combining the advantages of SunTag and tandem split green fluorescent protein (GFP) in amplifying fluorescent signals, dSaCas9-tdTomato10x and dSpCas9-GFP14x were constructed to enable efficient two-color CASVIT detection. Real-time two-color imaging indicates that replication compartments (RCs) frequently come into contact with each other but do not mix, suggesting that RC territory is highly stable. Last, two-color CASVIT enables simultaneous tracking of viral DNA and host chromatin, which reveals that a dramatic loss of telomeric and centromeric DNA occurs in host cells at the early stage of viral replication. Overall, our work has established a framework for developing CRISPR-Cas9-based imaging tools to study DNA viruses in living cells. IMPORTANCE Herpes simplex virus 1 (HSV-1), a representative of the family Herpesviridae, is a ubiquitous pathogen that can establish lifelong infections and widely affects human health. Viral infection is a dynamic process that involves many steps and interactions with various cellular structures, including host chromatin. A common viral replication strategy is to form RCs that concentrate factors required for viral replication. Efficient strategies for imaging the dynamics of viral genomes, RC formation, and the interaction between the virus and host offer the opportunity to dissect the steps of the infection process and determine the mechanism underlying each step. We have developed an efficient two-color imaging system based on CRISPR-Cas9 technology to detect HSV-1 genomes quantitatively in living cells. Our results shed light on novel aspects of RC dynamics and virus-host interactions.
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Herpes Simples , Herpesvirus Humano 1 , Interações entre Hospedeiro e Microrganismos , Replicação Viral , Humanos , Linhagem Celular , Cromatina , Herpes Simples/genética , Herpesvirus Humano 1/genética , Interações entre Hospedeiro e Microrganismos/genética , Replicação Viral/genética , DNA Viral/análise , DNA Viral/genéticaRESUMO
Herpes simplex virus 2 (HSV-2) establishes latent infection in dorsal root ganglion (DRG) neurons after productive (lytic) infection in peripheral tissues. A neuron-specific microRNA, miR-138, favors HSV-1 latency by repressing viral ICP0 and host Oct-1 and Foxc1 genes, yet the role of miR-138 in HSV-2 infection was unknown. The ICP0 mRNAs of HSV-1, HSV-2, and chimpanzee herpesvirus each have one to two canonical miR-138 binding sites. The sites are 100% conserved in 308 HSV-1 and 300 HSV-2 published sequences of clinical isolates. In cotransfection assays, miR-138 repressed HSV-2 ICP0 expression through the seed region and surrounding interactions that are different from HSV-1. An HSV-2 mutant with disrupted miR-138 binding sites on ICP0 showed increased ICP0 expression in Neuro-2a cells. Photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation confirmed miR-138 binding to HSV-2 ICP0 and identified UL19 and UL20 as additional targets whose expression was repressed by miR-138 during cotransfection. In Neuro-2a cells, transfected miR-138 and its antagomir decreased and increased HSV-2 replication, respectively, and a knockout experiment showed that miR-138's host targets OCT-1 and FOXC1 were important for HSV-2 replication. In primary mouse DRG neurons, both ICP0 and FOXC1 positively regulated HSV-2 replication, but both overexpressed and endogenous miR-138 suppressed HSV-2 replication primarily by repressing ICP0 expression. Thus, miR-138 can suppress HSV-2 neuronal replication through multiple viral and host pathways. These results reveal functional similarities and mechanistic differences in how miR-138 regulates HSV-1 and HSV-2 infection and indicate an evolutionary advantage of using miR-138 to repress lytic infection in neurons. IMPORTANCE HSV-1 and HSV-2 are closely related viruses with major differences. Both viruses establish latency in neurons from which they reactivate to cause disease. A key aspect of HSV latency is repression of productive infection in neurons. Based on previous work with HSV-1, we investigated the role of a neuron-specific microRNA, miR-138, in HSV-2 infection and established it as a repressor of HSV-2 productive infection in neuronal cells. This repression is mediated mainly by targeting viral ICP0 and host Foxc1 mRNAs, but other pathways also contribute. Despite functional conservation of the role of miR-138 between HSV-1 and HSV-2, many molecular mechanisms differ, including how miR-138 represses ICP0 expression and miR-138 targeting of HSV-2 but not HSV-1 UL19 and UL20. To our knowledge, this study provides the first example of host microRNA regulation of HSV-2 infection.
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Herpes Simples , Herpesvirus Humano 2 , MicroRNAs , Neurônios , Animais , Fatores de Transcrição Forkhead , Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Camundongos , MicroRNAs/genética , Neurônios/virologia , Fator 1 de Transcrição de Octâmero , Ubiquitina-Proteína Ligases/metabolismo , Latência Viral/genética , Replicação ViralRESUMO
Enteroviruses (EVs) are common causes of viral encephalitis in children. To better understand the epidemiological and pathological characteristics of EV encephalitis, we enrolled suspected encephalitis patients younger than 15 years old in Hangzhou, China, from October 2016 to September 2019 for cerebrospinal fluid (CSF) collection and analyses. A total of 7735 CSF samples were collected, among which 330 (4.27%) were positive for the EV genome. The positivity rate was significantly higher in boys than girls (χ2 = â 5.68, p = â 0.02). The monthly case numbers peaked from June to August (80.30%). Among the different age groups, the 0-2 months age group showed the highest number of cases (28.48% of all cases). The 6-7 years (10.82%) and 9-10 years (9.29%) age groups showed the highest EV-positivity rates among suspected encephalitis cases. Sixty-two EV-positive and 53 control CSF samples were collected for Bio-Plex Pro human cytokine assays that simultaneously tested 48 cytokines. Principle component analyses showed significant separation between EV-positive and control samples, but insignificant separation between children and newborns. The levels of 28 cytokines and chemokines were significantly elevated in the EV-positive group including many proinflammatory and a few anti-inflammatory cytokines, as well as chemokines belonging to the CC and CXC subfamilies. Only one cytokine, stem cell growth factor-ß, showed a decrease in the EV-positive group. Thus, this study revealed age, sex, and seasonal preferences for EV encephalitis incidences in children and identified many cytokines dysregulated during EV encephalitis.
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Encefalite Viral , Encefalite , Infecções por Enterovirus , Enterovirus , Adolescente , Líquido Cefalorraquidiano , Criança , China/epidemiologia , Citocinas , Encefalite Viral/epidemiologia , Enterovirus/genética , Infecções por Enterovirus/epidemiologia , Feminino , Humanos , Lactente , Recém-Nascido , MasculinoRESUMO
Enteroviruses (EVs) are major causes of viral meningoencephalitis in children. To better understand the pathogenesis and identify potential biomarkers, cerebrospinal fluid proteome in children (n = 52) suffering from EV meningoencephalitis was compared to that in EV-negative control subjects (n = 53) using the BoxCar acquisition technique. Among 1697 proteins identified, 1193 with robust assay readouts were used for quantitative analyses. Differential expression analyses identified 154 upregulated and 227 downregulated proteins in the EV-positive group. Functional analyses showed that the upregulated proteins are mainly related to activities of lymphocytes and cytokines, inflammation, and responses to stress and viral invasion, while the downregulated proteins are mainly related to neuronal integrity and activity as well as neurogenesis. According to receiver operating characteristic analysis results, Rho-GDP-dissociation inhibitor 2 exhibited the highest sensitivity (96.2%) and specificity (100%) for discriminating EV-positive from EV-negative patients. The chemokine CXCL10 was most upregulated (>300-fold) with also high sensitivity (92.3%) and specificity (94.3%) for indicating EV positivity. Thus, this study uncovered perturbations of multiple host processes due to EV meningoencephalitis, especially the general trend of enhanced immune responses but impaired neuronal functions. The identified dysregulated proteins may also prompt biomarker development.
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Infecções por Enterovirus , Enterovirus , Meningoencefalite , Biomarcadores , Líquido Cefalorraquidiano , Criança , Enterovirus/genética , Infecções por Enterovirus/diagnóstico , Infecções por Enterovirus/genética , Humanos , Meningoencefalite/diagnóstico , ProteômicaRESUMO
Herpes simplex virus (HSV), including HSV-1 and HSV-2, is an important pathogen that can cause many diseases. Usually these diseases are recurrent and incurable. After lytic infection on the surface of peripheral mucosa, HSV can enter sensory neurons and establish latent infection during which viral replication ceases. Moreover, latent virus can re-enter the replication cycle by reactivation and return to peripheral tissues to start recurrent infection. This ability to escape host immune surveillance during latent infection and to spread during reactivation is a viral survival strategy and the fundamental reason why no drug can completely eradicate the virus at present. Although there are many studies on latency and reactivation of HSV, and much progress has been made, many specific mechanisms of the process remain obscure or even controversial due to the complexity of this process and the limitations of research models. This paper reviews the major results of research on HSV latency and reactivation, and discusses future research directions in this field.
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Herpes Simples , Herpesvirus Humano 1 , Ativação Viral , Latência Viral , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Humanos , Ativação Viral/fisiologia , Latência Viral/fisiologia , Replicação ViralRESUMO
Herpes simplex virus 1 (HSV-1) latency entails the repression of productive ("lytic") gene expression. An attractive hypothesis to explain some of this repression involves inhibition of the expression of ICP0, a lytic gene activator, by a viral microRNA, miR-H2, which is completely complementary to ICP0 mRNA. To test this hypothesis, we engineered mutations that disrupt miR-H2 without affecting ICP0 in HSV-1. The mutant virus exhibited drastically reduced expression of miR-H2 but showed wild-type levels of infectious virus production and no increase in ICP0 expression in lytically infected cells, which is consistent with the weak expression of miR-H2 relative to the level of ICP0 mRNA in that setting. Following corneal inoculation of mice, the mutant was not significantly different from wild-type virus in terms of infectious virus production in the trigeminal ganglia during acute infection, mouse mortality, or the rate of reactivation from explanted latently infected ganglia. Critically, the mutant was indistinguishable from wild-type virus for the expression of ICP0 and other lytic genes in acutely and latently infected mouse trigeminal ganglia. The latter result may be related to miR-H2 being less effective in inhibiting ICP0 expression in transfection assays than a host microRNA, miR-138, which has previously been shown to inhibit lytic gene expression in infected ganglia by targeting ICP0 mRNA. Additionally, transfected miR-138 reduced lytic gene expression in infected cells more effectively than miR-H2. While this study provides little support for the hypothesis that miR-H2 promotes latency by inhibiting ICP0 expression, the possibility remains that miR-H2 might target other genes during latency. IMPORTANCE: Herpes simplex virus 1 (HSV-1), which causes a variety of diseases, can establish lifelong latent infections from which virus can reactivate to cause recurrent disease. Latency is the most biologically interesting and clinically vexing feature of the virus. Ever since miR-H2's discovery as a viral microRNA bearing complete sequence complementarity to the mRNA for the important viral gene activator ICP0, inhibition of ICP0 expression by miR-H2 has been a major hypothesis to help explain the repression of lytic gene expression during latency. However, this hypothesis remained untested in latently infected animals. Using a miR-H2-deficient mutant virus, we found no evidence that miR-H2 represses the expression of ICP0 or other lytic genes in cells or mice infected with HSV-1. Although miR-H2 can repress ICP0 expression in transfection assays, such repression is weak. The results suggest that other mechanisms for miR-H2 activity and for the repression of lytic gene expression during latency deserve investigation.
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Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/genética , MicroRNAs/genética , Mutação , RNA Viral/genética , Gânglio Trigeminal/virologia , Ubiquitina-Proteína Ligases/genética , Ativação Viral , Animais , Sítios de Ligação , Linhagem Celular , Modelos Animais de Doenças , Herpes Simples/mortalidade , Herpes Simples/virologia , Humanos , Proteínas Imediatamente Precoces/química , Proteínas Imediatamente Precoces/metabolismo , Camundongos , Interferência de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Virulência , Latência Viral/genética , Replicação ViralRESUMO
Ribosomal frameshifting entails slippage of the translational machinery during elongation. Frameshifting permits expression of more than one polypeptide from an otherwise monocistronic mRNA, and can restore expression of polypeptides in the face of frameshift mutations. A common mutation conferring acyclovir resistance in patients with herpes simplex virus disease deletes one cytosine from a run of six cytosines (C-chord) in the viral thymidine kinase (tk) gene. However, this mutation does not abolish TK activity, which is important for pathogenicity. To investigate how this mutant retains TK activity, we engineered and analyzed viruses expressing epitope-tagged TK. We found that the mutant's TK activity can be accounted for by low levels of full-length TK polypeptide produced by net -1 frameshifting during translation. The efficiency of frameshifting was relatively high, 3-5%, as the polypeptide from the reading frame generated by the deletion, which lacks stop codons (nonstop), was poorly expressed mainly because of inefficient protein synthesis. Stop codons introduced into this reading frame greatly increased its expression, but greatly decreased the level of full-length TK, indicating that frameshifting is strongly stimulated by a new mechanism, nonstop mRNA, which we hypothesize involves stalling of ribosomes on the polyA tail. Mutational studies indicated that frameshifting occurs on or near the C-chord, a region lacking a canonical slippery sequence. Nonstop stimulation of frameshifting also occurred when the C-chord was replaced with a canonical slippery sequence from HIV. This mechanism thus permits biologically and clinically relevant TK synthesis, and may occur more generally.
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Farmacorresistência Viral/genética , Mudança da Fase de Leitura do Gene Ribossômico/genética , Herpesvirus Humano 1/enzimologia , Proteínas Oncogênicas/genética , Timidina Quinase/genética , Aciclovir , Animais , Autorradiografia , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Chlorocebus aethiops , Cromossomos Artificiais Bacterianos/genética , Primers do DNA/genética , Mutação da Fase de Leitura/genética , Herpesvirus Humano 1/patogenicidade , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Plasmídeos/genética , Reação em Cadeia da Polimerase em Tempo Real , Timidina Quinase/metabolismo , Células VeroRESUMO
We investigated thymidine kinase (tk) mutants isolated during multiple episodes of recurrent bilateral acyclovir resistant herpes simplex keratitis in an immunocompetent patient. From one eye, we found a single guanine insertion, previously shown to greatly reduce TK expression, and from the other, a previously unidentified substitution, which genetic experiments confirmed confers drug resistance. The substitution, although distant from substrate binding sites, reduced thymidine phosphorylation 10-20-fold, and acyclovir phosphorylation >100-fold. This phenotype should permit reactivation from latency to cause recurrent disease. The results may have implications for the prevalence and prevention of acyclovir resistance in patients with herpes simplex keratitis.
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Aciclovir/farmacologia , Antivirais/farmacologia , Farmacorresistência Viral , Herpesvirus Humano 1/efeitos dos fármacos , Ceratite Herpética/virologia , Adulto , Substituição de Aminoácidos , DNA Viral/química , DNA Viral/genética , Herpesvirus Humano 1/isolamento & purificação , Humanos , Masculino , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Fosforilação , Mutação Puntual , Recidiva , Análise de Sequência de DNA , Timidina/metabolismo , Timidina Quinase/genética , Timidina Quinase/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Herpes simplex virus 1 (HSV-1) latent infection entails repression of viral lytic genes in neurons. By functional screening using luciferase-expressing HSV-1, we identify ten neuron-specific microRNAs potentially repressing HSV-1 neuronal replication. Transfection of miR-9, the most active candidate from the screen, decreases HSV-1 replication and gene expression in Neuro-2a cells. Ectopic expression of miR-9 from lentivirus or recombinant HSV-1 suppresses HSV-1 replication in male primary mouse neurons in culture and mouse trigeminal ganglia in vivo, and reactivation from latency in the primary neurons. Target prediction and validation identify transcription factors Oct-1, a known co-activator of HSV transcription, and all three Onecut family members as miR-9 targets. Knockdown of ONECUT2 decreases HSV-1 yields in Neuro-2a cells. Overexpression of each ONECUT protein increases HSV-1 replication in Neuro-2a cells, human induced pluripotent stem cell-derived neurons, and primary mouse neurons, and accelerates reactivation from latency in the mouse neurons. Mutagenesis, ChIP-seq, RNA-seq, ChIP-qPCR and ATAC-seq results suggest that ONECUT2 can nonspecifically bind to viral genes via its CUT domain, globally stimulate viral gene transcription, reduce viral heterochromatin and enhance the accessibility of viral chromatin. Thus, neuronal miR-9 promotes viral epigenetic silencing and latency by targeting multiple host transcription factors important for lytic gene activation.
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Herpes Simples , Herpesvirus Humano 1 , Células-Tronco Pluripotentes Induzidas , MicroRNAs , Humanos , Masculino , Animais , Camundongos , Herpesvirus Humano 1/genética , MicroRNAs/genética , Neurônios , Herpes Simples/genética , Fatores de Transcrição , Epigênese Genética , Proteínas de HomeodomínioRESUMO
To be clinically relevant, drug-resistant mutants must both evade drug action and retain pathogenicity. Many acyclovir-resistant herpes simplex virus mutants from clinical isolates have one or two base insertions (G8 and G9) or one base deletion (G6) in a homopolymeric run of seven guanines (G string) in the gene encoding thymidine kinase (TK). Nevertheless, G8 and G9 mutants express detectable TK activity and can reactivate from latency in mice, a pathogenicity marker. On the basis of studies using cell-free systems, ribosomal frameshifting can explain this ability to express TK. To investigate frameshifting in infected cells, we constructed viruses that express epitope-tagged versions of wild-type and mutant TKs. We measured TK activity by plaque autoradiography and expression of frameshifted and unframeshifted TK polypeptides using a very sensitive immunoprecipitation-Western blotting method. The G6 mutant expressed â¼0.01% of wild-type levels of TK polypeptide. For the G9 mutant, consistent with previous results, much TK expression could be ascribed to reversion. For the G8 mutant, from these assays and pulse-labeling studies, we determined the ratio of synthesis of frameshifted to unframeshifted polypeptides to be 1:100. The effects of stop codons before or after the G string argue that frameshifting can initiate within the first six guanines. However, frameshifting efficiency was altered by stop codons downstream of the string in the 0 frame. The G8 mutant expressed only 0.1% of the wild-type level of full-length TK, considerably lower than estimated previously. Thus, remarkably low levels of TK are sufficient for reactivation from latency in mice.
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Aciclovir/farmacologia , Antivirais/farmacologia , Mutação , Simplexvirus/genética , Timidina Quinase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Códon sem Sentido , Farmacorresistência Viral/genética , Mutação da Fase de Leitura , Deleção de Genes , Expressão Gênica , Ordem dos Genes , Humanos , Mutagênese Insercional , Estabilidade Proteica , Simplexvirus/metabolismo , Timidina Quinase/metabolismoRESUMO
OBJECTIVE: This study investigated the proteomic characteristics of cerebrospinal fluid (CSF) in patients with varicella zoster virus (VZV) meningitis to understanding the pathogenesis of central nervous system (CNS) infection by reactivated VZV. METHOD: We used data-independent acquisition model to analyze the CSF proteomic differences of 28 patients with VZV meningitis and 11 herpes zoster (HZ) patients. According to the clinical manifestations at discharge, 28 VZV meningitis patients were divided into favorable outcome group and unfavorable outcome (UO) group and their differences in CSF proteome were also analyzed. RESULTS: Compared with the HZ group, the proteins (CXCL10, ELANE, IL-1RN, MPO, PRTN3, etc.) related to inflammation and immune cell activation were significantly upregulated in the VZV meningitis group (p < .01). The protein related to the nerve function and energy metabolism (CKMT1B, SLITRK3, Synaptotagmin-3, KIF5B, etc.) were significantly downregulated (p < .05). The levels of a pro-inflammatory factor, IL-18, in CSF were significantly higher in patients in the UO group as compared to patients with favorable prognosis (p < .05). CONCLUSION: Inflammatory immune response is an important pathophysiological mechanism of CNS infection by VZV, and the CSF IL-18 levels might be a potential prognostic indicator of the outcomes of VZV meningitis.
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Herpes Zoster , Meningite , Humanos , Herpesvirus Humano 3/fisiologia , Interleucina-18 , Proteômica , ProteínasRESUMO
Oncolytic viruses (OVs) encoding various transgenes are being evaluated for cancer immunotherapy. Diverse factors such as cytokines, immune checkpoint inhibitors, tumor-associated antigens, and T cell engagers have been exploited as transgenes. These modifications are primarily aimed to reverse the immunosuppressive tumor microenvironment. By contrast, antiviral restriction factors that inhibit the replication of OVs and result in suboptimal oncolytic activity have received far less attention. Here, we report that guanylate-binding protein 1 (GBP1) is potently induced during HSV-1 infection and restricts HSV-1 replication. Mechanistically, GBP1 remodels cytoskeletal organization to impede nuclear entry of HSV-1 genome. Previous studies have established that IpaH9.8, a bacterial E3 ubiquitin ligase, targets GBPs for proteasomal degradation. We therefore engineered an oncolytic HSV-1 to express IpaH9.8 and found that the modified OV effectively antagonized GBP1, replicated to a higher titer in vitro and showed superior antitumor activity in vivo. Our study features a strategy for improving the replication of OVs via targeting a restriction factor and achieving promising therapeutic efficacy.
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Stimulator of interferon genes (STING) orchestrates the production of proinflammatory cytokines in response to cytosolic double-stranded DNA; however, the pathophysiological significance and molecular mechanism underlying the folding and maturation of nascent STING protein at the endoplasmic reticulum (ER) remain unknown. Here we report that the SEL1L-HRD1 protein complex-the most conserved branch of ER-associated degradation (ERAD)-is a negative regulator of the STING innate immunity by ubiquitinating and targeting nascent STING protein for proteasomal degradation in the basal state. SEL1L or HRD1 deficiency in macrophages specifically amplifies STING signalling and immunity against viral infection and tumour growth. Mechanistically, nascent STING protein is a bona fide substrate of SEL1L-HRD1 in the basal state, uncoupled from ER stress or its sensor inositol-requiring enzyme 1α. Hence, our study not only establishes a key role of SEL1L-HRD1 ERAD in innate immunity by limiting the size of the activable STING pool, but identifies a regulatory mechanism and therapeutic approach to targeting STING.
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Degradação Associada com o Retículo Endoplasmático , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/metabolismo , Proteínas/metabolismo , Retículo Endoplasmático/metabolismo , Imunidade InataRESUMO
Herpesviruses are ubiquitous human pathogens. After productive (lytic) infection, all human herpesviruses are able to establish life-long latent infection and reactivate from it. Latent infection entails suppression of viral replication, maintenance of the viral genome in infected cells, and the ability to reactivate. Most human herpesviruses encode microRNAs (miRNAs) that regulate these processes during latency. Meanwhile, cellular miRNAs are hijacked by herpesviruses to participate in these processes. The viral or cellular miRNAs either directly target viral transcripts or indirectly affect viral infection through host pathways. These findings shed light on the molecular determinants that control the lytic-latent switch and may lead to novel therapeutics targeting latent infection. We discuss the multiple mechanisms by which miRNAs regulate herpesvirus latency, focusing on the patterns in these mechanisms.
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Herpesvirus Humano 8 , Infecção Latente , MicroRNAs , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Latência Viral/genética , Replicação ViralRESUMO
Purpose: A series of complications caused by severe COVID-19 can significantly affect short-term results. Therefore, early diagnosis is essential for critically COVID-19 patients. we aimed to investigate the correlation among D-dimer levels, lymphocyte subsets, cytokines, and disease severity in COVID-19 patients. Methods: Systematic review and meta- analysis of PubMed, Scopus, Web of Science, Cochrane Central Register of Controlled Trials, Embase, clinical trials, and China National Knowledge Infrastructure (CNKI) until 1 August 2022. We considered case-control, and cohort studies that compared laboratory parameters between patients with severe or non-serious diseases or between survivors and non-survivors. Pooled data was assessed by use of a random-effects model and used I 2 to test heterogeneity. We assessed the risk of bias using the Newcastle- Ottawa Scale. Results: Of the 5,561 identified studies, 32 were eligible and included in our analysis (N = 3,337 participants). Random-effect results indicated that patients with COVID-19 in severe group had higher levels for D-dimer (WMD = 1.217 mg/L, 95%CI=[0.788, 1.646], P < 0.001), neutrophil-to-lymphocyte ratio (NLR) (WMD = 6.939, 95%CI = [4.581, 9.297], P < 0.001), IL-2 (WMD = 0.371 pg/ml, 95%CI = [-0.190, 0.932], P = 0.004), IL-4 (WMD = 0.139 pg/ml, 95%CI = [0.060, 0.219], P = 0.717), IL-6 (WMD = 44.251 pg/ml, 95%CI = [27.010, 61.493], P < 0.001), IL-10 (WMD = 3.718 pg/ml, 95%CI = [2.648, 4.788], P < 0.001) as well as lower levels of lymphocytes (WMD = -0.468( × 109/L), 95%CI = [-0.543, -0.394], P < 0.001), T cells (WMD = -446.746(/µL), 95%CI = [-619.607, -273.885], P < 0.001), B cells (WMD = -60.616(/µL), 95%CI = [-96.452, -24.780], P < 0.001), NK cells (WMD = -68.297(/µL), 95%CI = [-90.600, -45.994], P < 0.001), CD3+T cells (WMD = -487.870(/µL), 95%CI = [-627.248, -348.492], P < 0.001), CD4+T cells (WMD = -290.134(/µL), 95%CI = [-370.834, -209.435], P < 0.001), CD8+T cells (WMD = -188.781(/µL), 95%CI = [-227.806, -149.757], P < 0.001). Conclusions: There is a correlation among higher levels of D-dimer, cytokines, lower levels of lymphocyte subsets, and disease severity in COVID-19 patients. These effective biomarkers may help clinicians to evaluate the severity and prognosis of COVID-19. This study is registered with PROSPERO, number CRD42020196659. Systematic review registration: https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=196659; PROSPERO registration number: CRD42020196659.
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Background and Aims: Antithrombin (AT) is the most important physiological inhibitor in vivo, and coagulation factor II (FII) or prothrombin is a coagulation factor vital to life. The purpose of our research was to illustrate the connection between gene mutations and the corresponding deficiencies of AT and FII. Methods: Functional and molecular analyses were performed. The possible impact of the mutation was analyzed by online bioinformatics software. ClustalX-2.1-win and PyMol/Swiss-Pdb Viewer software were used for conservative analyses and to generate molecular graphic images, respectively. Results: The proband showed a lower limb venous thrombosis and acute pulmonary embolism infarction with reduced AT activity (50%). His mother, with subcutaneous ecchymosis, had reduced activities of AT and FII, of 44 and 5%, respectively. Molecular analysis showed that both the proband and his mother carried c.964A > T (p.Lys322stop) heterozygotes in SERPINC1. The difference was that his mother carried homozygous c.494C > T (p.Thr165Met) in F2, while the proband was wild type. Bioinformatics and model analysis indicated that mutations may destroy the function and structure of AT and FII protein. Conclusion: This study identified a novel mutation of SERPINC1 and a missense mutation of F2, which may be the molecular mechanism leading to AT and FII deficiency in this family. It will help genetic diagnosis and counseling for thrombotic families.
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Herpes simplex virus 1 (HSV-1) can productively infect multiple cell types and establish latent infection in neurons. Infected cell protein 0 (ICP0) is an HSV-1 E3 ubiquitin ligase crucial for productive infection and reactivation from latency. However, our knowledge about its targets especially in neuronal cells is limited. We confirmed that, like in non-neuronal cells, ICP0-null virus exhibited major replication defects in primary mouse neurons and Neuro-2a cells. We identified many ICP0-interacting proteins in Neuro-2a cells, 293T cells, and human foreskin fibroblasts by mass spectrometry-based interactome analysis. Co-immunoprecipitation assays validated ICP0 interactions with acyl-coenzyme A thioesterase 8 (ACOT8), complement C1q binding protein (C1QBP), ovarian tumour domain-containing protein 4 (OTUD4), sorting nexin 9 (SNX9), and vimentin (VIM) in both Neuro-2a and 293T cells. Overexpression and knockdown experiments showed that SNX9 restricted replication of an ICP0-null but not wild-type virus in Neuro-2a cells. Ubiquitinome analysis by immunoprecipitating the trypsin-digested ubiquitin reminant followed by mass spectrometry identified numerous candidate ubiquitination substrates of ICP0 in infected Neuro-2a cells, among which OTUD4 and VIM were novel substrates confirmed to be ubiquitinated by transfected ICP0 in Neuro-2a cells despite no evidence of their degradation by ICP0. Expression of OTUD4 was induced independently of ICP0 during HSV-1 infection. Overexpressed OTUD4 enhanced type I interferon expression during infection with the ICP0-null but not wild-type virus. In summary, by combining two proteomic approaches followed by confirmatory and functional experiments, we identified and validated multiple novel targets of ICP0 and revealed potential restrictive activities of SNX9 and OTUD4 in neuronal cells.
RESUMO
Herpes simplex virus 1 (HSV-1) expresses a large number of miRNAs, and their function is still not completely understood. In addition, HSV-1 has been found to deregulate host miRNAs, which adds to the complexity of the regulation of efficient virus replication. In this study, we comprehensively addressed the deregulation of host miRNAs by massive-parallel sequencing. We found that only miRNAs expressed from a single cluster, miR-183/96/182, are reproducibly deregulated during productive infection. These miRNAs are predicted to regulate a great number of potential targets involved in different cellular processes and have only 33 shared targets. Among these, members of the FoxO family of proteins were identified as potential targets for all three miRNAs. However, our study shows that the upregulated miRNAs do not affect the expression of FoxO proteins, moreover, these proteins were upregulated in HSV-1 infection. Furthermore, we show that the individual FoxO proteins are not required for efficient HSV-1 replication. Taken together, our results indicate a complex and redundant response of infected cells to the virus infection that is efficiently inhibited by the virus.
Assuntos
Herpes Simples , Herpesvirus Humano 1 , MicroRNAs , Herpes Simples/genética , Herpesvirus Humano 1/fisiologia , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Regulação para Cima , Replicação ViralRESUMO
We describe an optimized procedure for replacing the dihydrouridine residues of charged tRNAs with Cy3 and Cy5 dyes linked to a hydrazide group, and demonstrate that the labeled molecules are functional in ribosomal activities including 30S initiation complex formation, EF-Tu-dependent binding to the ribosome, translocation, and polypeptide synthesis. This procedure should be straightforwardly generalizable to the incorporation of other hydrazide-linked fluorophores into tRNA or other dihydrouridine-containing RNAs. In addition, we use a rapid turnover FRET experiment, measuring energy transfer between Cy5-labeled tRNA(fMet) and Cy3-labeled fMetPhe-tRNA(Phe), to obtain direct evidence supporting the hypothesis that the early steps of translocation involve movements of the flexible 3'-single-stranded regions of the tRNAs, with the considerable increase in the distance separating the two tRNA tertiary cores occurring later in the process.