RESUMEN
Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.
Asunto(s)
Encefalopatías Metabólicas Innatas/genética , Tronco Encefálico/metabolismo , Tronco Encefálico/virología , ARN/química , ARN/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Encefalopatías Metabólicas Innatas/patología , Tronco Encefálico/patología , Encefalitis Viral/genética , Escherichia coli/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/virología , Herpesvirus Humano 1 , Humanos , Interferones/metabolismo , Intrones/genética , Masculino , Ratones , Proteínas Mutantes/metabolismo , Mutación/genética , Sistemas de Lectura Abierta/genética , Linaje , ARN Nucleotidiltransferasas/química , ARN Nucleotidiltransferasas/deficiencia , ARN Nucleotidiltransferasas/genética , Receptor Toll-Like 3/metabolismo , Replicación ViralRESUMEN
Most cases of herpes simplex virus 1 (HSV-1) encephalitis (HSE) remain unexplained1,2. Here, we report on two unrelated people who had HSE as children and are homozygous for rare deleterious variants of TMEFF1, which encodes a cell membrane protein that is preferentially expressed by brain cortical neurons. TMEFF1 interacts with the cell-surface HSV-1 receptor NECTIN-1, impairing HSV-1 glycoprotein D- and NECTIN-1-mediated fusion of the virus and the cell membrane, blocking viral entry. Genetic TMEFF1 deficiency allows HSV-1 to rapidly enter cortical neurons that are either patient specific or derived from CRISPR-Cas9-engineered human pluripotent stem cells, thereby enhancing HSV-1 translocation to the nucleus and subsequent replication. This cellular phenotype can be rescued by pretreatment with type I interferon (IFN) or the expression of exogenous wild-type TMEFF1. Moreover, ectopic expression of full-length TMEFF1 or its amino-terminal extracellular domain, but not its carboxy-terminal intracellular domain, impairs HSV-1 entry into NECTIN-1-expressing cells other than neurons, increasing their resistance to HSV-1 infection. Human TMEFF1 is therefore a host restriction factor for HSV-1 entry into cortical neurons. Its constitutively high abundance in cortical neurons protects these cells from HSV-1 infection, whereas inherited TMEFF1 deficiency renders them susceptible to this virus and can therefore underlie HSE.
Asunto(s)
Encéfalo , Encefalitis por Herpes Simple , Herpesvirus Humano 1 , Proteínas de la Membrana , Internalización del Virus , Animales , Femenino , Humanos , Masculino , Encéfalo/citología , Encéfalo/metabolismo , Encéfalo/virología , Encefalitis por Herpes Simple/virología , Encefalitis por Herpes Simple/metabolismo , Herpesvirus Humano 1/patogenicidad , Herpesvirus Humano 1/fisiología , Homocigoto , Interferón Tipo I/metabolismo , Interferón Tipo I/inmunología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Nectinas/genética , Nectinas/metabolismo , Neuronas/citología , Neuronas/metabolismo , Neuronas/virología , Células Madre Pluripotentes/citología , Replicación Viral , Preescolar , Adulto Joven , LinajeRESUMEN
Inborn errors of TLR3-dependent type I IFN immunity in cortical neurons underlie forebrain herpes simplex virus-1 (HSV-1) encephalitis (HSE) due to uncontrolled viral growth and subsequent cell death. We report an otherwise healthy patient with HSE who was compound heterozygous for nonsense (R422*) and frameshift (P493fs9*) RIPK3 variants. Receptor-interacting protein kinase 3 (RIPK3) is a ubiquitous cytoplasmic kinase regulating cell death outcomes, including apoptosis and necroptosis. In vitro, the R422* and P493fs9* RIPK3 proteins impaired cellular apoptosis and necroptosis upon TLR3, TLR4, or TNFR1 stimulation and ZBP1/DAI-mediated necroptotic cell death after HSV-1 infection. The patient's fibroblasts displayed no detectable RIPK3 expression. After TNFR1 or TLR3 stimulation, the patient's cells did not undergo apoptosis or necroptosis. After HSV-1 infection, the cells supported excessive viral growth despite normal induction of antiviral IFN-ß and IFN-stimulated genes (ISGs). This phenotype was, nevertheless, rescued by application of exogenous type I IFN. The patient's human pluripotent stem cell (hPSC)-derived cortical neurons displayed impaired cell death and enhanced viral growth after HSV-1 infection, as did isogenic RIPK3-knockout hPSC-derived cortical neurons. Inherited RIPK3 deficiency therefore confers a predisposition to HSE by impairing the cell death-dependent control of HSV-1 in cortical neurons but not their production of or response to type I IFNs.
Asunto(s)
Encefalitis por Herpes Simple , Herpes Simple , Herpesvirus Humano 1 , Humanos , Muerte Celular , Encefalitis por Herpes Simple/genética , Herpesvirus Humano 1/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Receptores Tipo I de Factores de Necrosis Tumoral , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismoRESUMEN
Enterovirus (EV) infection rarely results in life-threatening infection of the central nervous system. We report two unrelated children with EV30 and EV71 rhombencephalitis. One patient carries compound heterozygous TLR3 variants (loss-of-function F322fs2* and hypomorphic D280N), and the other is homozygous for an IFIH1 variant (loss-of-function c.1641+1G>C). Their fibroblasts respond poorly to extracellular (TLR3) or intracellular (MDA5) poly(I:C) stimulation. The baseline (TLR3) and EV-responsive (MDA5) levels of IFN-ß in the patients' fibroblasts are low. EV growth is enhanced at early and late time points of infection in TLR3- and MDA5-deficient fibroblasts, respectively. Treatment with exogenous IFN-α2b before infection renders both cell lines resistant to EV30 and EV71, whereas post-infection treatment with IFN-α2b rescues viral susceptibility fully only in MDA5-deficient fibroblasts. Finally, the poly(I:C) and viral phenotypes of fibroblasts are rescued by the expression of WT TLR3 or MDA5. Human TLR3 and MDA5 are critical for cell-intrinsic immunity to EV, via the control of baseline and virus-induced type I IFN production, respectively.
Asunto(s)
Encefalitis Viral/inmunología , Infecciones por Enterovirus/inmunología , Helicasa Inducida por Interferón IFIH1/genética , Receptor Toll-Like 3/genética , Células Cultivadas , Preescolar , Encefalitis Viral/genética , Enterovirus/efectos de los fármacos , Enterovirus/fisiología , Infecciones por Enterovirus/genética , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Fibroblastos/virología , Humanos , Lactante , Interferón alfa-2/farmacología , Helicasa Inducida por Interferón IFIH1/inmunología , Interferón beta/inmunología , Interferón beta/metabolismo , Mutación con Pérdida de Función , Masculino , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/inmunología , Poli I-C/farmacología , Rombencéfalo/virología , Receptor Toll-Like 3/inmunología , Replicación Viral/efectos de los fármacosRESUMEN
Inborn errors of TLR3-dependent IFN-α/ß- and IFN-λ-mediated immunity in the CNS can underlie herpes simplex virus 1 (HSV-1) encephalitis (HSE). The respective contributions of IFN-α/ß and IFN-λ are unknown. We report a child homozygous for a genomic deletion of the entire coding sequence and part of the 3'-UTR of the last exon of IFNAR1, who died of HSE at the age of 2 years. An older cousin died following vaccination against measles, mumps, and rubella at 12 months of age, and another 17-year-old cousin homozygous for the same variant has had other, less severe, viral illnesses. The encoded IFNAR1 protein is expressed on the cell surface but is truncated and cannot interact with the tyrosine kinase TYK2. The patient's fibroblasts and EBV-B cells did not respond to IFN-α2b or IFN-ß, in terms of STAT1, STAT2, and STAT3 phosphorylation or the genome-wide induction of IFN-stimulated genes. The patient's fibroblasts were susceptible to viruses, including HSV-1, even in the presence of exogenous IFN-α2b or IFN-ß. HSE is therefore a consequence of inherited complete IFNAR1 deficiency. This viral disease occurred in natural conditions, unlike those previously reported in other patients with IFNAR1 or IFNAR2 deficiency. This experiment of nature indicates that IFN-α/ß are essential for anti-HSV-1 immunity in the CNS.
Asunto(s)
Encefalitis por Herpes Simple , Herpesvirus Humano 1/metabolismo , Receptor de Interferón alfa y beta/deficiencia , Adolescente , Preescolar , Encefalitis por Herpes Simple/genética , Encefalitis por Herpes Simple/metabolismo , Encefalitis por Herpes Simple/patología , Células HEK293 , Herpesvirus Humano 1/genética , Humanos , Interferones/genética , Interferones/metabolismo , Masculino , Receptor de Interferón alfa y beta/metabolismoRESUMEN
Human herpes simplex virus 1 (HSV-1) encephalitis can be caused by inborn errors of the TLR3 pathway, resulting in impairment of CNS cell-intrinsic antiviral immunity. Deficiencies of the TLR3 pathway impair cell-intrinsic immunity to vesicular stomatitis virus (VSV) and HSV-1 in fibroblasts, and to HSV-1 in cortical but not trigeminal neurons. The underlying molecular mechanism is thought to involve impaired IFN-α/ß induction by the TLR3 recognition of dsRNA viral intermediates or by-products. However, we show here that human TLR3 controls constitutive levels of IFNB mRNA and secreted bioactive IFN-ß protein, and thereby also controls constitutive mRNA levels for IFN-stimulated genes (ISGs) in fibroblasts. Tlr3-/- mouse embryonic fibroblasts also have lower basal ISG levels. Moreover, human TLR3 controls basal levels of IFN-ß secretion and ISG mRNA in induced pluripotent stem cell-derived cortical neurons. Consistently, TLR3-deficient human fibroblasts and cortical neurons are vulnerable not only to both VSV and HSV-1, but also to several other families of viruses. The mechanism by which TLR3 restricts viral growth in human fibroblasts and cortical neurons in vitro and, by inference, by which the human CNS prevents infection by HSV-1 in vivo, is therefore based on the control of early viral infection by basal IFN-ß immunity.
Asunto(s)
Corteza Cerebral/inmunología , Fibroblastos/inmunología , Herpesvirus Humano 1/inmunología , Interferón beta/inmunología , Neuronas/inmunología , Receptor Toll-Like 3/inmunología , Vesiculovirus/inmunología , Animales , Línea Celular , Corteza Cerebral/patología , Corteza Cerebral/virología , Fibroblastos/patología , Fibroblastos/virología , Humanos , Interferón beta/genética , Ratones , Ratones Noqueados , Neuronas/patología , Neuronas/virología , Receptor Toll-Like 3/genéticaRESUMEN
Autosomal recessive IRF7 and IRF9 deficiencies impair type I and III IFN immunity and underlie severe influenza pneumonitis. We report three unrelated children with influenza A virus (IAV) infection manifesting as acute respiratory distress syndrome (IAV-ARDS), heterozygous for rare TLR3 variants (P554S in two patients and P680L in the third) causing autosomal dominant (AD) TLR3 deficiency. AD TLR3 deficiency can underlie herpes simplex virus-1 (HSV-1) encephalitis (HSE) by impairing cortical neuron-intrinsic type I IFN immunity to HSV-1. TLR3-mutated leukocytes produce normal levels of IFNs in response to IAV. In contrast, TLR3-mutated fibroblasts produce lower levels of IFN-ß and -λ, and display enhanced viral susceptibility, upon IAV infection. Moreover, the patients' iPSC-derived pulmonary epithelial cells (PECs) are susceptible to IAV. Treatment with IFN-α2b or IFN-λ1 rescues this phenotype. AD TLR3 deficiency may thus underlie IAV-ARDS by impairing TLR3-dependent, type I and/or III IFN-mediated, PEC-intrinsic immunity. Its clinical penetrance is incomplete for both IAV-ARDS and HSE, consistent with their typically sporadic nature.
Asunto(s)
Gripe Humana/genética , Patrón de Herencia/genética , Neumonía/genética , Receptor Toll-Like 3/deficiencia , Alelos , Niño , Preescolar , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Resultado Fatal , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Heterocigoto , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Lactante , Recién Nacido , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/fisiología , Interferones/metabolismo , Mutación con Pérdida de Función/genética , Pulmón/patología , Masculino , Mutación Missense/genética , Poli I-C/farmacología , Transporte de ProteínasRESUMEN
Herpes simplex virus-1 (HSV-1) encephalitis (HSE) is typically sporadic. Inborn errors of TLR3- and DBR1-mediated central nervous system cell-intrinsic immunity can account for forebrain and brainstem HSE, respectively. We report five unrelated patients with forebrain HSE, each heterozygous for one of four rare variants of SNORA31, encoding a small nucleolar RNA of the H/ACA class that are predicted to direct the isomerization of uridine residues to pseudouridine in small nuclear RNA and ribosomal RNA. We show that CRISPR/Cas9-introduced bi- and monoallelic SNORA31 deletions render human pluripotent stem cell (hPSC)-derived cortical neurons susceptible to HSV-1. Accordingly, SNORA31-mutated patient hPSC-derived cortical neurons are susceptible to HSV-1, like those from TLR3- or STAT1-deficient patients. Exogenous interferon (IFN)-ß renders SNORA31- and TLR3- but not STAT1-mutated neurons resistant to HSV-1. Finally, transcriptome analysis of SNORA31-mutated neurons revealed normal responses to TLR3 and IFN-α/ß stimulation but abnormal responses to HSV-1. Human SNORA31 thus controls central nervous system neuron-intrinsic immunity to HSV-1 by a distinctive mechanism.
Asunto(s)
Encefalitis por Herpes Simple/genética , Herpesvirus Humano 1/genética , Neuronas/inmunología , ARN Nucleolar Pequeño/genética , Adulto , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/virología , Preescolar , Encefalitis por Herpes Simple/inmunología , Encefalitis por Herpes Simple/patología , Encefalitis por Herpes Simple/virología , Femenino , Predisposición Genética a la Enfermedad , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/patogenicidad , Humanos , Inmunidad/genética , Lactante , Masculino , Metagenoma/genética , Metagenoma/inmunología , Persona de Mediana Edad , Neuronas/virología , ARN Nucleolar Pequeño/inmunologíaRESUMEN
Viruses build icosahedral capsids of specific size and shape by regulating the spatial arrangement of the hexameric and pentameric protein capsomers in the growing shell during assembly. In the T=7 capsids of Escherichia coli bacteriophage HK97 and other phages, 60 capsomers are hexons, while the rest are pentons that are correctly positioned during assembly. Assembly of the HK97 capsid to the correct size and shape has been shown to depend on specific ionic contacts between capsomers. We now describe additional ionic interactions within capsomers that also regulate assembly. Each is between the long hairpin, the "E-loop," that extends from one subunit to the adjacent subunit within the same capsomer. Glutamate E153 on the E-loop and arginine R210 on the adjacent subunit's backbone alpha-helix form salt bridges in hexamers and pentamers. Mutations that disrupt these salt bridges were lethal for virus production, because the mutant proteins assembled into tubes or sheets instead of capsids. X-ray structures show that the E153-R210 links are flexible and maintained during maturation despite radical changes in capsomer shape. The E153-R210 links appear to form early in assembly to enable capsomers to make programmed changes in their shape during assembly. The links also prevent flattening of capsomers and premature maturation. Mutant phenotypes and modeling support an assembly model in which flexible E153-R210 links mediate capsomer shape changes that control where pentons are placed to create normal-sized capsids. The E-loop may be conserved in other systems in order to play similar roles in regulating assembly.
Asunto(s)
Proteínas de la Cápside/metabolismo , Colifagos/fisiología , Multimerización de Proteína , Ensamble de Virus , Cápside/química , Cápside/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Unión ProteicaRESUMEN
We previously reported a series of de novo engineered cationic antibiotic peptides (eCAPs) consisting exclusively of arginine and tryptophan (WR) that display potent activity against diverse multidrug-resistant (MDR) bacterial strains. In this study, we sought to examine the influence of arginine compared to lysine on antibacterial properties by direct comparison of the WR peptides (8-18 residues) with a parallel series of engineered peptides containing only lysine and tryptophan. WR and WK series were compared for antibacterial activity by bacterial killing and growth inhibition assays and for mechanism of peptide-bacteria interactions by surface plasmon resonance and flow cytometry. Mammalian cytotoxicity was also assessed by flow cytometry, haemolytic and tetrazolium-based assays. The shortest arginine-containing peptides (8 and 10 mers) displayed a statistically significant increase in activity compared to the analogous lysine-containing peptides. The WR and WK peptides achieved maximum antibacterial activity at the 12-mer peptide (WK12 or WR12). Further examination of antibacterial mechanisms of the optimally active 12-mer peptides using surface plasmon resonance and flow cytometry demonstrates stronger interactions with Pseudomonasaeruginosa, greater membrane permeabilizing activity, and lower inhibitory effects of divalent cations on activity and membrane permeabilization properties of WR12 compared to WK12 (P < 0.05). Importantly, WK12 and WR12 displayed similar negligible haemolytic and cytotoxic effects at peptide concentrations up to ten times the MIC or 20 times the minimum bactericidal concentration. Thus, arginine, compared to lysine, can indeed yield enhanced antibacterial activity to minimize the required length to achieve functional antimicrobial peptides.