RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses.
Asunto(s)
COVID-19/metabolismo , Interacciones Huésped-Patógeno , Biosíntesis de Proteínas , Empalme del ARN , SARS-CoV-2/metabolismo , Proteínas no Estructurales Virales/metabolismo , Células A549 , Animales , COVID-19/virología , Chlorocebus aethiops , Células HEK293 , Humanos , Interferones/metabolismo , Transporte de Proteínas , ARN Mensajero/metabolismo , ARN Ribosómico 18S/metabolismo , ARN Citoplasmático Pequeño/química , ARN Citoplasmático Pequeño/metabolismo , Partícula de Reconocimiento de Señal/química , Partícula de Reconocimiento de Señal/metabolismo , Células Vero , Proteínas no Estructurales Virales/químicaRESUMEN
Spillover events of avian influenza A viruses (IAVs) to humans could represent the first step in a future pandemic1. Several factors that limit the transmission and replication of avian IAVs in mammals have been identified. There are several gaps in our understanding to predict which virus lineages are more likely to cross the species barrier and cause disease in humans1. Here, we identified human BTN3A3 (butyrophilin subfamily 3 member A3)2 as a potent inhibitor of avian IAVs but not human IAVs. We determined that BTN3A3 is expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts primarily at the early stages of the virus life cycle by inhibiting avian IAV RNA replication. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F or, rarely, 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, avian IAV serotypes, such as H7 and H9, that spilled over into humans also evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure3. Thus, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.
Asunto(s)
Aves , Interacciones Microbiota-Huesped , Virus de la Influenza A , Gripe Aviar , Gripe Humana , Zoonosis Virales , Animales , Humanos , Aves/virología , Virus de la Influenza A/clasificación , Virus de la Influenza A/genética , Virus de la Influenza A/crecimiento & desarrollo , Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/transmisión , Gripe Aviar/virología , Gripe Humana/prevención & control , Gripe Humana/transmisión , Gripe Humana/virología , Primates , Sistema Respiratorio/metabolismo , Sistema Respiratorio/virología , Medición de Riesgo , Zoonosis Virales/prevención & control , Zoonosis Virales/transmisión , Zoonosis Virales/virología , Replicación ViralRESUMEN
Interactions between viruses during coinfections can influence viral fitness and population diversity, as seen in the generation of reassortant pandemic influenza A virus (IAV) strains. However, opportunities for interactions between closely related viruses are limited by a process known as superinfection exclusion (SIE), which blocks coinfection shortly after primary infection. Using IAVs, we asked whether SIE, an effect which occurs at the level of individual cells, could limit interactions between populations of viruses as they spread across multiple cells within a host. To address this, we first measured the kinetics of SIE in individual cells by infecting them sequentially with 2 isogenic IAVs, each encoding a different fluorophore. By varying the interval between addition of the 2 IAVs, we showed that early in infection SIE does not prevent coinfection, but that after this initial lag phase the potential for coinfection decreases exponentially. We then asked how the kinetics of SIE onset controlled coinfections as IAVs spread asynchronously across monolayers of cells. We observed that viruses at individual coinfected foci continued to coinfect cells as they spread, because all new infections were of cells that had not yet established SIE. In contrast, viruses spreading towards each other from separately infected foci could only establish minimal regions of coinfection before reaching cells where coinfection was blocked. This created a pattern of separate foci of infection, which was recapitulated in the lungs of infected mice, and which is likely to be applicable to many other viruses that induce SIE. We conclude that the kinetics of SIE onset segregate spreading viral infections into discrete regions, within which interactions between virus populations can occur freely, and between which they are blocked.
Asunto(s)
Coinfección , Gripe Humana , Orthomyxoviridae , Sobreinfección , Ratones , Animales , Humanos , Virus ReordenadosRESUMEN
The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.
Asunto(s)
Vacunas contra la COVID-19 , COVID-19/diagnóstico , COVID-19/virología , Genética Inversa , SARS-CoV-2/genética , Células A549 , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Chlorocebus aethiops , Codón , Humanos , Hidrazonas/farmacología , Ratones , Morfolinas/farmacología , Sistemas de Lectura Abierta , Plásmidos/genética , Pirimidinas/farmacología , Serina Endopeptidasas/metabolismo , Células Vero , Proteínas Virales/metabolismoRESUMEN
Long noncoding RNAs (lncRNAs) are frequently associated with broad modulation of gene expression and thus provide the cell with the ability to synchronize entire metabolic processes. We used transcriptomic approaches to investigate whether the most abundant human cytomegalovirus-encoded lncRNA, RNA2.7, has this characteristic. By comparing cells infected with wild-type virus (WT) to cells infected with RNA2.7 deletion mutants, RNA2.7 was implicated in regulating a large number of cellular genes late in lytic infection. Pathway analysis indicated that >100 of these genes are associated with promoting cell movement, and the 10 most highly regulated of these were validated in further experiments. Morphological analysis and live cell tracking of WT- and RNA2.7 mutant-infected cells indicated that RNA2.7 is involved in promoting the movement and detachment of infected cells late in infection, and plaque assays using sparse cell monolayers indicated that RNA2.7 is also involved in promoting cell-to-cell spread of virus. Consistent with the observation that upregulated mRNAs are relatively A+U-rich, which is a trait associated with transcript instability, and that they are also enriched in motifs associated with mRNA instability, transcriptional inhibition experiments on WT- and RNA2.7 mutant-infected cells showed that four upregulated transcripts lived longer in the presence of RNA2.7. These findings demonstrate that RNA2.7 is required for promoting cell movement and viral spread late in infection and suggest that this may be due to general stabilization of A+U-rich transcripts. IMPORTANCE In addition to messenger RNAs (mRNAs), the human genome encodes a large number of long noncoding RNAs (lncRNAs). Many lncRNAs that have been studied in detail are associated with broad modulation of gene expression and have important biological roles. Human cytomegalovirus, which is a large, clinically important DNA virus, specifies four lncRNAs, one of which (RNA2.7) is expressed at remarkably high levels during lytic infection. Our studies show that RNA2.7 is required for upregulating a large number of human genes, about 100 of which are associated with cell movement, and for promoting the movement of infected cells and the spread of virus from one cell to another. Further bioinformatic and experimental analyses indicated that RNA2.7 may exert these effects by stabilizing mRNAs that are relatively rich in A and U nucleotides. These findings increase our knowledge of how human cytomegalovirus regulates the infected cell to promote its own success.
Asunto(s)
Citomegalovirus/genética , ARN Largo no Codificante/genética , Movimiento Celular/genética , Expresión Génica/genética , Regulación Viral de la Expresión Génica/genética , Humanos , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , ARN Viral/genética , Activación Transcripcional/genética , Transcriptoma , Regulación hacia Arriba , Replicación Viral/genéticaRESUMEN
Host innate immune defences play a critical role in restricting the intracellular propagation and pathogenesis of invading viral pathogens. Here we show that the histone H3.3 chaperone HIRA (histone cell cycle regulator) associates with promyelocytic leukaemia nuclear bodies (PML-NBs) to stimulate the induction of innate immune defences against herpes simplex virus 1 (HSV-1) infection. Following the activation of innate immune signalling, HIRA localized at PML-NBs in a Janus-Associated Kinase (JAK), Cyclin Dependent Kinase (CDK), and Sp100-dependent manner. RNA-seq analysis revealed that HIRA promoted the transcriptional upregulation of a broad repertoire of host genes that regulate innate immunity to HSV-1 infection, including those involved in MHC-I antigen presentation, cytokine signalling, and interferon stimulated gene (ISG) expression. ChIP-seq analysis revealed that PML, the principle scaffolding protein of PML-NBs, was required for the enrichment of HIRA onto ISGs, identifying a role for PML in the HIRA-dependent regulation of innate immunity to virus infection. Our data identifies independent roles for HIRA in the intrinsic silencing of viral gene expression and the induction of innate immune defences to restrict the initiation and propagation of HSV-1 infection, respectively. These intracellular host defences are antagonized by the HSV-1 ubiquitin ligase ICP0, which disrupts the stable recruitment of HIRA to infecting viral genomes and PML-NBs at spatiotemporally distinct phases of infection. Our study highlights the importance of histone chaperones to regulate multiple phases of intracellular immunity to virus infection, findings that are likely to be highly pertinent in the cellular restriction of many clinically important viral pathogens.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Fibroblastos/inmunología , Infecciones por Herpesviridae/inmunología , Herpesvirus Humano 1/patogenicidad , Chaperonas de Histonas/metabolismo , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata/inmunología , Factores de Transcripción/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Fibroblastos/citología , Fibroblastos/virología , Regulación Viral de la Expresión Génica , Infecciones por Herpesviridae/metabolismo , Infecciones por Herpesviridae/virología , Chaperonas de Histonas/genética , Humanos , Factores de Transcripción/genética , Replicación ViralRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1006769.].
RESUMEN
Detection of viral nucleic acids plays a critical role in the induction of intracellular host immune defences. However, the temporal recruitment of immune regulators to infecting viral genomes remains poorly defined due to the technical difficulties associated with low genome copy-number detection. Here we utilize 5-Ethynyl-2'-deoxyuridine (EdU) labelling of herpes simplex virus 1 (HSV-1) DNA in combination with click chemistry to examine the sequential recruitment of host immune regulators to infecting viral genomes under low multiplicity of infection conditions. Following viral genome entry into the nucleus, PML-nuclear bodies (PML-NBs) rapidly entrapped viral DNA (vDNA) leading to a block in viral replication in the absence of the viral PML-NB antagonist ICP0. This pre-existing intrinsic host defence to infection occurred independently of the vDNA pathogen sensor IFI16 (Interferon Gamma Inducible Protein 16) and the induction of interferon stimulated gene (ISG) expression, demonstrating that vDNA entry into the nucleus alone is not sufficient to induce a robust innate immune response. Saturation of this pre-existing intrinsic host defence during HSV-1 ICP0-null mutant infection led to the stable recruitment of PML and IFI16 into vDNA complexes associated with ICP4, and led to the induction of ISG expression. This induced innate immune response occurred in a PML-, IFI16-, and Janus-Associated Kinase (JAK)-dependent manner and was restricted by phosphonoacetic acid, demonstrating that vDNA polymerase activity is required for the robust induction of ISG expression during HSV-1 infection. Our data identifies dual roles for PML in the sequential regulation of intrinsic and innate immunity to HSV-1 infection that are dependent on viral genome delivery to the nucleus and the onset of vDNA replication, respectively. These intracellular host defences are counteracted by ICP0, which targets PML for degradation from the outset of nuclear infection to promote vDNA release from PML-NBs and the onset of HSV-1 lytic replication.
Asunto(s)
Regulación Viral de la Expresión Génica/efectos de los fármacos , Herpes Simple/metabolismo , Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno , Cuerpos de Inclusión Viral/metabolismo , Proteína de la Leucemia Promielocítica/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Virales/metabolismo , Línea Celular , Línea Celular Transformada , Células Cultivadas , Química Clic , Eliminación de Gen , Herpes Simple/tratamiento farmacológico , Herpes Simple/patología , Herpes Simple/virología , Herpesvirus Humano 1/crecimiento & desarrollo , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Inmunidad Innata/efectos de los fármacos , Cuerpos de Inclusión Viral/efectos de los fármacos , Cuerpos de Inclusión Viral/patología , Cuerpos de Inclusión Viral/virología , Cinética , Lisogenia/efectos de los fármacos , Mutación , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteína de la Leucemia Promielocítica/antagonistas & inhibidores , Proteína de la Leucemia Promielocítica/genética , Interferencia de ARN , Inhibidores de la Transcriptasa Inversa/farmacología , Ubiquitina-Proteína Ligasas/genética , Proteínas Virales/genética , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Babesia venatorum is an increasingly prominent zoonotic parasite that predominantly infects wild deer. Our molecular examination of Babesia infecting mammals in the United Kingdom identified 18S sequences in domestic sheep isolates identical to zoonotic B. venatorum. Identification of this parasite in livestock raises concerns for public health and farming policy in Europe.
Asunto(s)
Babesia/clasificación , Babesiosis/epidemiología , Babesiosis/parasitología , Especificidad del Huésped , Zoonosis/epidemiología , Zoonosis/parasitología , Animales , Babesia/genética , Reacción en Cadena de la Polimerasa , Vigilancia en Salud Pública , ARN Protozoario , ARN Ribosómico 18S/genética , Ovinos , Reino Unido/epidemiologíaRESUMEN
Canine degenerative myelopathy (DM) is a progressive neurological disorder that may be considered to be a large animal model for specific forms of the fatal human disease, familial amyotrophic lateral sclerosis (fALS). DM is associated with a c118G>A mutation of the superoxide dismutase 1 (Sod1) gene, and a significant proportion of cases are inherited in an autosomal recessive manner in contrast to the largely, but not exclusively, dominant mode of inheritance in fALS. The consensus view is that these Sod1/SOD1 mutations result in a toxic gain of function but the mechanisms remain unclear. Here we used an in vitro neuroblastoma cell line transfection system to monitor wild-type and mutant forms of SOD1 fusion proteins containing either a Cherry or an enhanced green fluorescent protein (EGFP) tag. These fusion proteins retained SOD1 enzymatic activity on a native gel assay system. We demonstrate that SOD1 aggregate density is significantly higher in DM transfectants compared to wild-type. In addition, we show by co-immunoprecipitation and confocal microscopy, evidence for a potential interaction between wild-type and mutant forms of SOD1 in co-transfected cells. While in vitro studies have shown SOD1 heterodimer formation in fALS models, this is the first report for DM SOD1. Therefore, despite for the majority of cases there is a difference in the mode of inheritance between fALS and DM, a similar interaction between wild-type and mutant SOD1 forms can occur. Clarifying the role of SOD1 in DM may also be of benefit to understanding the role of SOD1 in fALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Mutación/genética , Superóxido Dismutasa-1/genética , Superóxido Dismutasa/genética , Animales , Línea Celular , Modelos Animales de Enfermedad , Perros , Proteínas Fluorescentes Verdes/genética , Humanos , Enfermedades de la Médula Espinal/genéticaRESUMEN
Influenza viruses exhibit striking variations in particle morphology between strains. Clinical isolates of influenza A virus have been shown to produce long filamentous particles while laboratory-adapted strains are predominantly spherical. However, the role of the filamentous phenotype in the influenza virus infectious cycle remains undetermined. We used cryo-electron tomography to conduct the first three-dimensional study of filamentous virus ultrastructure in particles budding from infected cells. Filaments were often longer than 10 microns and sometimes had bulbous heads at their leading ends, some of which contained tubules we attribute to M1 while none had recognisable ribonucleoprotein (RNP) and hence genome segments. Long filaments that did not have bulbs were infrequently seen to bear an ordered complement of RNPs at their distal ends. Imaging of purified virus also revealed diverse filament morphologies; short rods (bacilliform virions) and longer filaments. Bacilliform virions contained an ordered complement of RNPs while longer filamentous particles were narrower and mostly appeared to lack this feature, but often contained fibrillar material along their entire length. The important ultrastructural differences between these diverse classes of particles raise the possibility of distinct morphogenetic pathways and functions during the infectious process.
Asunto(s)
Subtipo H3N2 del Virus de la Influenza A/ultraestructura , Virión/ultraestructura , Animales , Microscopía por Crioelectrón/métodos , Perros , Subtipo H3N2 del Virus de la Influenza A/fisiología , Células de Riñón Canino Madin Darby , Virión/fisiologíaRESUMEN
Herpesviruses are ubiquitous pathogens that cause a wide range of disease. Upon nuclear entry, their genomes associate with histones and chromatin modifying enzymes that regulate the progression of viral transcription and outcome of infection. While the composition and modification of viral chromatin has been extensively studied on bulk populations of infected cells by chromatin immunoprecipitation, this key regulatory process remains poorly defined at single-genome resolution. Here we use high-resolution quantitative imaging to investigate the spatial proximity of canonical and variant histones at individual Herpes Simplex Virus 1 (HSV-1) genomes within the first 90 minutes of infection. We identify significant population heterogeneity in the stable enrichment and spatial proximity of canonical histones (H2A, H2B, H3.1) at viral DNA (vDNA) relative to established promyelocytic leukaemia nuclear body (PML-NB) host factors that are actively recruited to viral genomes upon nuclear entry. We show the replication-independent histone H3.3/H4 chaperone Daxx to cooperate with PML to mediate the enrichment and spatial localization of variant histone H3.3 at vDNA that limits the rate of HSV-1 genome decompaction to restrict the progress of immediate-early (IE) transcription. This host response is counteracted by the viral ubiquitin ligase ICP0, which degrades PML to disperse Daxx and variant histone H3.3 from vDNA to stimulate the progression of viral genome expansion, IE transcription, and onset of HSV-1 replication. Our data support a model of intermediate and sequential histone assembly initiated by Daxx that limits the rate of HSV-1 genome decompaction independently of the stable enrichment of histones H2A and H2B at vDNA required to facilitate canonical nucleosome assembly. We identify HSV-1 genome decompaction upon nuclear infection to play a key role in the initiation and functional outcome of HSV-1 lytic infection, findings pertinent to the transcriptional regulation of many nuclear replicating herpesvirus pathogens.
RESUMEN
Respiratory syncytial virus (RSV) is an important human pathogen. Its nucleocapsid (NC), which comprises the negative sense RNA viral genome coated by the viral nucleoprotein N, is a critical assembly that serves as template for both mRNA synthesis and genome replication. We have previously described the X-ray structure of an NC-like structure: a decameric ring formed of N-RNA that mimics one turn of the helical NC. In the absence of experimental data we had hypothesized that the NC helix would be right-handed, as the N-N contacts in the ring appeared to more easily adapt to that conformation. We now unambiguously show that the RSV NC is a left-handed helix. We further show that the contacts in the ring can be distorted to maintain key N-N-protein interactions in a left-handed helix, and discuss the implications of the resulting atomic model of the helical NC for viral replication and transcription.
Asunto(s)
Nucleocápside/química , Nucleoproteínas/química , ARN Viral/química , Virus Sincitial Respiratorio Humano/química , Humanos , Modelos Moleculares , Nucleoproteínas/metabolismo , Unión Proteica , Conformación Proteica , ARN Viral/metabolismoRESUMEN
T cell responses against infections and cancer are directed by conventional dendritic cells (cDCs) in lymph nodes distant from the site of challenge. Migratory cDCs, which travel from the tissue to the lymph node, not only drive initial T cell activation but also transfer antigen to lymph node-resident cDCs. These resident cells have essential roles defining the character of the resulting T cell response; however, it is unknown how they can appropriately process and present antigens to suitably direct responses given their spatial separation. Here, using a novel strain of influenza A and a modified melanoma model, we show that tissue and lymph node cDC activation is harmonized and that this is driven by cotransfer of contextual cues. In the tumor, incomplete cDC activation in the tumor microenvironment is mirrored by lymph node-resident cDCs, whereas during influenza infection, pathogen-associated molecular patterns cotransferred with antigen drive TLR signaling in resident cDCs and their subsequent robust activation. This cotransfer mechanism explains how individual antigens can be handled distinctly by resident cDCs and how signals driving poor tumoral cDC activation further impact the lymph node. Our findings clarify how tissue context dictates antigenic and, consequently, T cell fate in the lymph node.
Asunto(s)
Gripe Humana , Humanos , Células Dendríticas , Antígenos , Ganglios Linfáticos , Linfocitos TRESUMEN
Human African trypanosomiasis, or sleeping sickness, is caused by the protozoan parasite Trypanosoma brucei and induces profound reactivity of glial cells and neuroinflammation when the parasites colonise the central nervous system. However, the transcriptional and functional responses of the brain to chronic T. brucei infection remain poorly understood. By integrating single cell and spatial transcriptomics of the mouse brain, we identify that glial responses triggered by infection are readily detected in the proximity to the circumventricular organs, including the lateral and 3rd ventricle. This coincides with the spatial localisation of both slender and stumpy forms of T. brucei. Furthermore, in silico predictions and functional validations led us to identify a previously unknown crosstalk between homeostatic microglia and Cd138+ plasma cells mediated by IL-10 and B cell activating factor (BAFF) signalling. This study provides important insights and resources to improve understanding of the molecular and cellular responses in the brain during infection with African trypanosomes.
Asunto(s)
Parásitos , Trypanosoma brucei brucei , Tripanosomiasis Africana , Animales , Factor Activador de Células B , Encéfalo/parasitología , Humanos , Interleucina-10 , Ratones , Microglía , Células Plasmáticas , Transcriptoma , Trypanosoma brucei brucei/genética , Tripanosomiasis Africana/parasitologíaRESUMEN
Inherited genetic factors can influence the severity of COVID-19, but the molecular explanation underpinning a genetic association is often unclear. Intracellular antiviral defenses can inhibit the replication of viruses and reduce disease severity. To better understand the antiviral defenses relevant to COVID-19, we used interferon-stimulated gene (ISG) expression screening to reveal that 2'-5'-oligoadenylate synthetase 1 (OAS1), through ribonuclease L, potently inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We show that a common splice-acceptor single-nucleotide polymorphism (Rs10774671) governs whether patients express prenylated OAS1 isoforms that are membrane-associated and sense-specific regions of SARS-CoV-2 RNAs or if they only express cytosolic, nonprenylated OAS1 that does not efficiently detect SARS-CoV-2. In hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting that this antiviral defense is a major component of a protective antiviral response.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , COVID-19/genética , COVID-19/fisiopatología , ARN Bicatenario/metabolismo , ARN Viral/metabolismo , SARS-CoV-2/fisiología , Regiones no Traducidas 5' , Células A549 , Animales , COVID-19/enzimología , COVID-19/inmunología , Quirópteros/genética , Quirópteros/virología , Coronaviridae/enzimología , Coronaviridae/genética , Coronaviridae/fisiología , Endorribonucleasas/metabolismo , Humanos , Interferones/inmunología , Isoenzimas/genética , Isoenzimas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Polimorfismo de Nucleótido Simple , Prenilación de Proteína , ARN Bicatenario/química , ARN Bicatenario/genética , ARN Viral/química , ARN Viral/genética , Retroelementos , SARS-CoV-2/genética , Índice de Severidad de la Enfermedad , Replicación ViralRESUMEN
Members of the Paramyxoviridae such as measles, mumps, and parainfluenza viruses have pleomorphic, enveloped virions that contain negative-sense unsegmented RNA genomes. This is encapsidated by multiple copies of a viral nucleocapsid protein N to form a helical ribonucleoprotein complex (termed the nucleocapsid), which acts as the template for both transcription and replication. Structure analysis of these viruses has proven challenging, owing to disordered regions in important constituent proteins, conformational flexibility in the nucleocapsid and the pleomorphic nature of virus particles. We conducted a low-resolution ultrastructural analysis of Sendai virus, a prototype paramyxovirus, using cryo-electron tomography. Virions are highly variable in size, ranging approximately from 110 to 540 nm in diameter. Envelope glycoproteins are densely packed on the virion surface, while nucleocapsids are clearly resolved in the virion interior. Subtomogram segmentation and filament tracing allowed us to define the path of many nucleocapsids and in some cases to determine the number of putative genomes within a single virus particle. Our findings indicate that these viruses may contain between one and six copies of their genome per virion and that there is no discernible order to nucleocapsid packaging.
Asunto(s)
Genoma Viral , Virus Sendai/genética , Virus Sendai/ultraestructura , Animales , Embrión de Pollo , Tomografía con Microscopio Electrónico , Dosificación de Gen , Nucleocápside/química , Nucleocápside/genética , Nucleocápside/metabolismo , Nucleocápside/ultraestructura , Tamaño de la Partícula , Virus Sendai/química , Virus Sendai/fisiologíaRESUMEN
Cryo electron microscopy (cryo-EM), a key method for structure determination involves imaging purified material embedded in vitreous ice. Images are then computationally processed to obtain three-dimensional structures approaching atomic resolution. There is increasing interest in extending structural studies by cryo-EM into the cell, where biological structures and processes may be imaged in context. The limited penetrating power of electrons prevents imaging of thick specimens (> 500 nm) however. Cryo-sectioning methods employed to overcome this are technically challenging, subject to artefacts or involve specialised and costly equipment. Here we describe the first structure of herpesvirus capsids determined by sub-tomogram averaging from nuclei of eukaryotic cells, achieved by cryo-electron tomography (cryo-ET) of re-vitrified cell sections prepared using the Tokuyasu method. Our reconstructions confirm that the capsid associated tegument complex is present on capsids prior to nuclear egress. We demonstrate that this method is suited to both 3D structure determination and correlative light/electron microscopy, thus expanding the scope of cryogenic cellular imaging.
Asunto(s)
Cápside/ultraestructura , Núcleo Celular/ultraestructura , Herpesvirus Humano 1/ultraestructura , Virión/ultraestructura , Animales , Línea Celular , Cricetinae , Microscopía por Crioelectrón , Tomografía con Microscopio ElectrónicoRESUMEN
Arboviruses are pathogens of humans and animals. A better understanding of the interactions between these pathogens and the arthropod vectors, such as mosquitoes, that transmit them is necessary to develop novel control measures. A major antiviral pathway in the mosquito vector is the exogenous small interfering RNA (exo-siRNA) pathway, which is induced by arbovirus-derived double-stranded RNA in infected cells. Although recent work has shown the key role played by Argonaute-2 (Ago-2) and Dicer-2 (Dcr-2) in this pathway, the regulatory mechanisms that govern these pathways have not been studied in mosquitoes. Here, we show that the Domino ortholog p400 has antiviral activity against the alphavirus Semliki Forest virus (Togaviridae) both in Aedes aegypti-derived cells and in vivo Antiviral activity of p400 was also demonstrated against chikungunya virus (Togaviridae) and Bunyamwera virus (Peribunyaviridae) but not Zika virus (Flaviviridae). p400 was found to be expressed across mosquito tissues and regulated ago-2 but not dcr-2 transcript levels in A. aegypti mosquitoes. These findings provide novel insights into the regulation of an important aedine exo-siRNA pathway effector protein, Ago-2, by the Domino ortholog p400. They add functional insights to previous observations of this protein's antiviral and RNA interference regulatory activities in Drosophila melanogasterIMPORTANCE Female Aedes aegypti mosquitoes are vectors of human-infecting arthropod-borne viruses (arboviruses). In recent decades, the incidence of arthropod-borne viral infections has grown dramatically. Vector competence is influenced by many factors, including the mosquito's antiviral defenses. The exogenous small interfering RNA (siRNA) pathway is a major antiviral response restricting arboviruses in mosquitoes. While the roles of the effectors of this pathway, Argonaute-2 and Dicer-2 are well characterized, nothing is known about its regulation in mosquitoes. In this study, we demonstrate that A. aegypti p400, whose ortholog Domino in Drosophila melanogaster is a chromatin-remodeling ATPase member of the Tip60 complex, regulates siRNA pathway activity and controls ago-2 expression levels. In addition, we found p400 to have antiviral activity against different arboviruses. Therefore, our study provides new insights into the regulation of the antiviral response in A. aegypti mosquitoes.
Asunto(s)
Aedes/genética , Proteínas Argonautas/genética , Proteínas de Insectos/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Aedes/virología , Animales , Arbovirus/fisiología , Femenino , Regulación de la Expresión Génica , Mosquitos Vectores/genética , Mosquitos Vectores/virologíaRESUMEN
The obligate intracellular parasite Toxoplasma gondii replicates in an unusual process, described as internal budding. Multiple dausghter parasites are formed sequentially within a single mother cell, requiring replication and distribution of essential organelles such as micronemes. These organelles are thought to be formed de novo in the developing daughter cells. Using dual labelling of a microneme protein MIC2 and super-resolution microscopy, we show that micronemes are recycled from the mother to the forming daughter parasites using a highly dynamic F-actin network. While this recycling pathway is F-actin dependent, de novo synthesis of micronemes appears to be F-actin independent. The F-actin network connects individual parasites, supports long, multidirectional vesicular transport, and regulates transport, density and localisation of micronemal vesicles. The residual body acts as a storage and sorting station for these organelles. Our data describe an F-actin dependent mechanism in apicomplexans for transport and recycling of maternal organelles during intracellular development.