RESUMEN
B.1.351 is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant most resistant to antibody neutralization. We demonstrate how the dose and number of immunizations influence protection. Nonhuman primates received two doses of 30 or 100 µg of Moderna's mRNA-1273 vaccine, a single immunization of 30 µg, or no vaccine. Two doses of 100 µg of mRNA-1273 induced 50% inhibitory reciprocal serum dilution neutralizing antibody titers against live SARS-CoV-2 p.Asp614Gly and B.1.351 of 3,300 and 240, respectively. Higher neutralizing responses against B.1.617.2 were also observed after two doses compared to a single dose. After challenge with B.1.351, there was ~4- to 5-log10 reduction of viral subgenomic RNA and low to undetectable replication in bronchoalveolar lavages in the two-dose vaccine groups, with a 1-log10 reduction in nasal swabs in the 100-µg group. These data establish that a two-dose regimen of mRNA-1273 will be critical for providing upper and lower airway protection against major variants of concern.
Asunto(s)
Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , Primates/inmunología , SARS-CoV-2/inmunología , Vacuna nCoV-2019 mRNA-1273 , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/virología , Línea Celular , Chlorocebus aethiops , Femenino , Humanos , Macaca mulatta , Masculino , Mesocricetus , Primates/virología , ARN Viral/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunación/métodos , Células Vero , Carga Viral/métodosRESUMEN
Viral reproduction is contingent on viral protein synthesis that relies on the host ribosomes. As such, viruses have evolved remarkable strategies to hijack the host translational apparatus in order to favor viral protein production and to interfere with cellular innate defenses. Here, we describe the approaches viruses use to exploit the translation machinery, focusing on commonalities across diverse viral families, and discuss the functional relevance of this process. We illustrate the complementary strategies host cells utilize to block viral protein production and consider how cells ensure an efficient antiviral response that relies on translation during this tug of war over the ribosome. Finally, we highlight potential roles mRNA modifications and ribosome quality control play in translational regulation and innate immunity. We address these topics in the context of the COVID-19 pandemic and focus on the gaps in our current knowledge of these mechanisms, specifically in viruses with pandemic potential.
Asunto(s)
COVID-19 , Biosíntesis de Proteínas , Virosis , Virus , Humanos , COVID-19/genética , COVID-19/inmunología , Pandemias , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/inmunología , ARN Viral/genética , ARN Viral/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , Virosis/genética , Virosis/inmunología , Virus/genética , Virus/inmunología , Ribosomas/genética , Ribosomas/inmunología , Ribosomas/virologíaRESUMEN
The mechanisms by which human immunodeficiency virus 1 (HIV-1) avoids immune surveillance by dendritic cells (DCs), and thereby prevents protective adaptive immune responses, remain poorly understood. Here we showed that HIV-1 actively arrested antiviral immune responses by DCs, which contributed to efficient HIV-1 replication in infected individuals. We identified the RNA helicase DDX3 as an HIV-1 sensor that bound abortive HIV-1 RNA after HIV-1 infection and induced DC maturation and type I interferon responses via the signaling adaptor MAVS. Notably, HIV-1 recognition by the C-type lectin receptor DC-SIGN activated the mitotic kinase PLK1, which suppressed signaling downstream of MAVS, thereby interfering with intrinsic host defense during HIV-1 infection. Finally, we showed that PLK1-mediated suppression of DDX3-MAVS signaling was a viral strategy that accelerated HIV-1 replication in infected individuals.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Dendríticas/virología , Infecciones por VIH/inmunología , VIH-1/fisiología , Evasión Inmune , Inmunidad , Macrófagos/virología , Proteínas Adaptadoras Transductoras de Señales/genética , Extractos Celulares , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Estudios de Cohortes , ARN Helicasas DEAD-box/metabolismo , Células Dendríticas/inmunología , Regulación Viral de la Expresión Génica , Células HEK293 , Infecciones por VIH/virología , Interacciones Huésped-Patógeno/genética , Humanos , Interferón beta/sangre , Macrófagos/inmunología , Polimorfismo de Nucleótido Simple , ARN Viral/inmunología , ARN Viral/metabolismo , Receptores de Reconocimiento de Patrones/metabolismo , Transducción de Señal , Carga Viral/genéticaRESUMEN
Faithful maintenance of immune homeostasis relies on the capacity of the cellular immune surveillance machinery to recognize "nonself", such as the presence of pathogenic RNA. Several families of pattern-recognition receptors exist that detect immunostimulatory RNA and then induce cytokine-mediated antiviral and proinflammatory responses. Here, we review the distinct features of bona fide RNA sensors, Toll-like receptors and retinoic-acid inducible gene-I (RIG-I)-like receptors in particular, with a focus on their functional specificity imposed by cell-type-dependent expression, subcellular localization, and ligand preference. Furthermore, we highlight recent advances on the roles of nucleotide-binding oligomerization domain (NOD)-like receptors and DEAD-box or DEAH-box RNA helicases in an orchestrated RNA-sensing network and also discuss the relevance of RNA sensor polymorphisms in human disease.
Asunto(s)
Inmunidad Innata/inmunología , ARN Viral/inmunología , Receptores de Reconocimiento de Patrones/inmunología , Citocinas/inmunología , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/inmunología , Proteína 58 DEAD Box/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/inmunología , ARN Helicasas DEAD-box/metabolismo , Humanos , Interferón Tipo I/inmunología , Proteínas NLR/genética , Proteínas NLR/inmunología , Proteínas NLR/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Transducción de Señal/inmunología , Receptores Toll-Like/metabolismoRESUMEN
The deployment of effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to eradicate the coronavirus disease 2019 (COVID-19) pandemic. Many licensed vaccines confer protection by inducing long-lived plasma cells (LLPCs) and memory B cells (MBCs), cell types canonically generated during germinal center (GC) reactions. Here, we directly compared two vaccine platforms-mRNA vaccines and a recombinant protein formulated with an MF59-like adjuvant-looking for their abilities to quantitatively and qualitatively shape SARS-CoV-2-specific primary GC responses over time. We demonstrated that a single immunization with SARS-CoV-2 mRNA, but not with the recombinant protein vaccine, elicited potent SARS-CoV-2-specific GC B and T follicular helper (Tfh) cell responses as well as LLPCs and MBCs. Importantly, GC responses strongly correlated with neutralizing antibody production. mRNA vaccines more efficiently induced key regulators of the Tfh cell program and influenced the functional properties of Tfh cells. Overall, this study identifies SARS-CoV-2 mRNA vaccines as strong candidates for promoting robust GC-derived immune responses.
Asunto(s)
Anticuerpos Neutralizantes/metabolismo , Linfocitos B/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , Centro Germinal/inmunología , SARS-CoV-2/fisiología , Linfocitos T Colaboradores-Inductores/inmunología , Vacunas Sintéticas/inmunología , Antígenos Virales/genética , Antígenos Virales/inmunología , Células Cultivadas , Epítopos , Humanos , Activación de Linfocitos , Polisorbatos , ARN Viral/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Escualeno , Vacunación , Vacunas de ARNmRESUMEN
SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full-length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19.
Asunto(s)
Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/prevención & control , Pandemias/prevención & control , Neumonía Viral/prevención & control , ARN Mensajero/inmunología , ARN Viral/inmunología , Vacunas Virales/administración & dosificación , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/virología , Betacoronavirus/inmunología , Betacoronavirus/patogenicidad , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , COVID-19 , Vacunas contra la COVID-19 , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/patología , Modelos Animales de Enfermedad , Furina/genética , Furina/inmunología , Humanos , Inmunidad Humoral/efectos de los fármacos , Inmunización/métodos , Inmunogenicidad Vacunal , Memoria Inmunológica/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Nanopartículas/administración & dosificación , Nanopartículas/química , Neumonía Viral/inmunología , Neumonía Viral/patología , ARN Mensajero/genética , ARN Viral/genética , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Sintéticas , Vacunas Virales/biosíntesis , Vacunas Virales/genéticaRESUMEN
Cyclic oligonucleotide-based antiphage signalling systems (CBASS) protect prokaryotes from viral (phage) attack through the production of cyclic oligonucleotides, which activate effector proteins that trigger the death of the infected host1,2. How bacterial cyclases recognize phage infection is not known. Here we show that staphylococcal phages produce a structured RNA transcribed from the terminase subunit genes, termed CBASS-activating bacteriophage RNA (cabRNA), which binds to a positively charged surface of the CdnE03 cyclase and promotes the synthesis of the cyclic dinucleotide cGAMP to activate the CBASS immune response. Phages that escape the CBASS defence harbour mutations that lead to the generation of a longer form of the cabRNA that cannot activate CdnE03. As the mammalian cyclase OAS1 also binds viral double-stranded RNA during the interferon response, our results reveal a conserved mechanism for the activation of innate antiviral defence pathways.
Asunto(s)
Bacterias , Nucleotidiltransferasas , ARN Viral , Fagos de Staphylococcus , Animales , 2',5'-Oligoadenilato Sintetasa/metabolismo , Bacterias/enzimología , Bacterias/inmunología , Evolución Molecular , Inmunidad Innata , Nucleotidiltransferasas/metabolismo , Oligonucleótidos/inmunología , Oligonucleótidos/metabolismo , ARN Viral/inmunología , ARN Viral/metabolismo , Transducción de Señal/inmunología , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/inmunologíaRESUMEN
RNA viruses have evolved elaborate strategies to protect their genomes, including 5' capping. However, until now no RNA 5' cap has been identified for hepatitis C virus1,2 (HCV), which causes chronic infection, liver cirrhosis and cancer3. Here we demonstrate that the cellular metabolite flavin adenine dinucleotide (FAD) is used as a non-canonical initiating nucleotide by the viral RNA-dependent RNA polymerase, resulting in a 5'-FAD cap on the HCV RNA. The HCV FAD-capping frequency is around 75%, which is the highest observed for any RNA metabolite cap across all kingdoms of life4-8. FAD capping is conserved among HCV isolates for the replication-intermediate negative strand and partially for the positive strand. It is also observed in vivo on HCV RNA isolated from patient samples and from the liver and serum of a human liver chimeric mouse model. Furthermore, we show that 5'-FAD capping protects RNA from RIG-I mediated innate immune recognition but does not stabilize the HCV RNA. These results establish capping with cellular metabolites as a novel viral RNA-capping strategy, which could be used by other viruses and affect anti-viral treatment outcomes and persistence of infection.
Asunto(s)
Flavina-Adenina Dinucleótido , Hepacivirus , Caperuzas de ARN , ARN Viral , Animales , Humanos , Ratones , Quimera/virología , Flavina-Adenina Dinucleótido/metabolismo , Hepacivirus/genética , Hepacivirus/inmunología , Hepatitis C/virología , Reconocimiento de Inmunidad Innata , Hígado/virología , Estabilidad del ARN , ARN Viral/química , ARN Viral/genética , ARN Viral/inmunología , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Replicación Viral/genética , Caperuzas de ARN/metabolismoRESUMEN
As long-lived post-mitotic cells, neurons employ unique strategies to resist pathogen infection while preserving cellular function. Here, using a murine model of Zika virus (ZIKV) infection, we identified an innate immune pathway that restricts ZIKV replication in neurons and is required for survival upon ZIKV infection of the central nervous system (CNS). We found that neuronal ZIKV infection activated the nucleotide sensor ZBP1 and the kinases RIPK1 and RIPK3, core components of virus-induced necroptotic cell death signaling. However, activation of this pathway in ZIKV-infected neurons did not induce cell death. Rather, RIPK signaling restricted viral replication by altering cellular metabolism via upregulation of the enzyme IRG1 and production of the metabolite itaconate. Itaconate inhibited the activity of succinate dehydrogenase, generating a metabolic state in neurons that suppresses replication of viral genomes. These findings demonstrate an immunometabolic mechanism of viral restriction during neuroinvasive infection.
Asunto(s)
Glicoproteínas/metabolismo , Hidroliasas/metabolismo , Neuronas/fisiología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Receptores de Reconocimiento de Patrones/metabolismo , Infección por el Virus Zika/inmunología , Virus Zika/fisiología , Animales , Muerte Celular , Células Cultivadas , Modelos Animales de Enfermedad , Glicoproteínas/genética , Humanos , Hidroliasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuroprotección , ARN Viral/inmunología , Proteínas de Unión al ARN , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Succinato Deshidrogenasa/metabolismo , Succinatos/metabolismo , Replicación ViralRESUMEN
Recognition of viral RNA by the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiates innate antiviral immune response. How the binding of viral RNA to and activation of the RLRs are regulated remains enigmatic. In this study, we identified ZCCHC3 as a positive regulator of the RLRs including RIG-I and MDA5. ZCCHC3 deficiency markedly inhibited RNA virus-triggered induction of downstream antiviral genes, and ZCCHC3-deficient mice were more susceptible to RNA virus infection. ZCCHC3 was associated with RIG-I and MDA5 and functions in two distinct processes for regulation of RIG-I and MDA5 activities. ZCCHC3 bound to dsRNA and enhanced the binding of RIG-I and MDA5 to dsRNA. ZCCHC3 also recruited the E3 ubiquitin ligase TRIM25 to the RIG-I and MDA5 complexes to facilitate its K63-linked polyubiquitination and activation. Thus, ZCCHC3 is a co-receptor for RIG-I and MDA5, which is critical for RLR-mediated innate immune response to RNA virus.
Asunto(s)
Proteína 58 DEAD Box/metabolismo , Infecciones por Virus ARN/inmunología , Virus ARN/fisiología , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteínas de Unión al ADN/metabolismo , Regulación Viral de la Expresión Génica , Células HEK293 , Humanos , Inmunidad Innata , Helicasa Inducida por Interferón IFIH1/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica , ARN Viral/inmunología , Proteínas de Unión al ARN/genética , Células THP-1 , Factores de Transcripción/metabolismo , UbiquitinaciónRESUMEN
Upon sensing viral RNA, mammalian RIG-I-like receptors (RLRs) activate downstream signals using caspase activation and recruitment domains (CARDs), which ultimately promote transcriptional immune responses that have been well studied. In contrast, the downstream signaling mechanisms for invertebrate RLRs are much less clear. For example, the Caenorhabditis elegans RLR DRH-1 lacks annotated CARDs and up-regulates the distinct output of RNA interference. Here, we found that similar to mammal RLRs, DRH-1 signals through two tandem CARDs (2CARD) to induce a transcriptional immune response. Expression of DRH-1(2CARD) alone in the intestine was sufficient to induce immune gene expression, increase viral resistance, and promote thermotolerance, a phenotype previously associated with immune activation in C. elegans. We also found that DRH-1 is required in the intestine to induce immune gene expression, and we demonstrate subcellular colocalization of DRH-1 puncta with double-stranded RNA inside the cytoplasm of intestinal cells upon viral infection. Altogether, our results reveal mechanistic and spatial insights into antiviral signaling in C. elegans, highlighting unexpected parallels in RLR signaling between C. elegans and mammals.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Transducción de Señal , Animales , Caenorhabditis elegans/inmunología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/inmunología , Transducción de Señal/inmunología , Intestinos/inmunología , Intestinos/virología , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , ARN Bicatenario/metabolismo , ARN Bicatenario/inmunología , Inmunidad Innata , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , ARN Viral/inmunología , ARN Viral/metabolismo , ARN Viral/genéticaRESUMEN
Intron-containing RNA expressed from the HIV-1 provirus activates type 1 interferon in primary human blood cells, including CD4+ T cells, macrophages, and dendritic cells. To identify the innate immune receptor required for detection of intron-containing RNA expressed from the HIV-1 provirus, a loss-of-function screen was performed with short hairpin RNA-expressing lentivectors targeting twenty-one candidate genes in human monocyte-derived dendritic cells. Among the candidate genes tested, only knockdown of XPO1 (CRM1), IFIH1 (MDA5), or MAVS prevented activation of the interferon-stimulated gene ISG15. The importance of IFIH1 protein was demonstrated by rescue of the knockdown with nontargetable IFIH1 coding sequence. Inhibition of HIV-1-induced ISG15 by the IFIH1-specific Nipah virus V protein, and by IFIH1-transdominant 2-CARD domain-deletion or phosphomimetic point mutations, indicates that IFIH1 (MDA5) filament formation, dephosphorylation, and association with MAVS are all required for innate immune activation in response to HIV-1 transduction. Since both IFIH1 (MDA5) and DDX58 (RIG-I) signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation. RNA-Seq showed that IFIH1 knockdown in dendritic cells globally disrupted the induction of IFN-stimulated genes by HIV-1. Finally, specific enrichment of unspliced HIV-1 RNA by IFIH1 (MDA5), over two orders of magnitude, was revealed by formaldehyde cross-linking immunoprecipitation (f-CLIP). These results demonstrate that IFIH1 is the innate immune receptor for intron-containing RNA from the HIV-1 provirus and that IFIH1 potentially contributes to chronic inflammation in people living with HIV-1, even in the presence of effective antiretroviral therapy.
Asunto(s)
Células Dendríticas , VIH-1 , Inmunidad Innata , Helicasa Inducida por Interferón IFIH1 , Intrones , Provirus , ARN Viral , Humanos , VIH-1/genética , VIH-1/inmunología , Helicasa Inducida por Interferón IFIH1/genética , Helicasa Inducida por Interferón IFIH1/metabolismo , Provirus/genética , Células Dendríticas/inmunología , Células Dendríticas/virología , Células Dendríticas/metabolismo , Intrones/genética , ARN Viral/genética , ARN Viral/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Infecciones por VIH/genética , Carioferinas/genética , Carioferinas/metabolismoRESUMEN
Detailed understanding of the signaling intermediates that confer the sensing of intracellular viral nucleic acids for induction of type I interferons is critical for strategies to curtail viral mechanisms that impede innate immune defenses. Here we show that the activation of the microtubule-associated guanine nucleotide exchange factor GEF-H1, encoded by Arhgef2, is essential for sensing of foreign RNA by RIG-I-like receptors. Activation of GEF-H1 controls RIG-I-dependent and Mda5-dependent phosphorylation of IRF3 and induction of IFN-ß expression in macrophages. Generation of Arhgef2(-/-) mice revealed a pronounced signaling defect that prevented antiviral host responses to encephalomyocarditis virus and influenza A virus. Microtubule networks sequester GEF-H1 that upon activation is released to enable antiviral signaling by intracellular nucleic acid detection pathways.
Asunto(s)
Inmunidad Innata/inmunología , Microtúbulos/inmunología , ARN Viral/inmunología , Factores de Intercambio de Guanina Nucleótido Rho/inmunología , Transducción de Señal/inmunología , Animales , Células COS , Chlorocebus aethiops , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/inmunología , ARN Helicasas DEAD-box/metabolismo , Expresión Génica/inmunología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Inmunidad Innata/genética , Immunoblotting , Virus de la Influenza A/genética , Factor 3 Regulador del Interferón/inmunología , Factor 3 Regulador del Interferón/metabolismo , Helicasa Inducida por Interferón IFIH1 , Interferón beta/genética , Interferón beta/inmunología , Interferón beta/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microtúbulos/metabolismo , Fosforilación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal/genéticaRESUMEN
RNA interference (RNAi) functions as a potent antiviral immunity in plants and invertebrates; however, whether RNAi plays antiviral roles in mammals remains unclear. Here, using human enterovirus 71 (HEV71) as a model, we showed HEV71 3A protein as an authentic viral suppressor of RNAi during viral infection. When the 3A-mediated RNAi suppression was impaired, the mutant HEV71 readily triggered the production of abundant HEV71-derived small RNAs with canonical siRNA properties in cells and mice. These virus-derived siRNAs were produced from viral dsRNA replicative intermediates in a Dicer-dependent manner and loaded into AGO, and they were fully active in degrading cognate viral RNAs. Recombinant HEV71 deficient in 3A-mediated RNAi suppression was significantly restricted in human somatic cells and mice, whereas Dicer deficiency rescued HEV71 infection independently of type I interferon response. Thus, RNAi can function as an antiviral immunity, which is induced and suppressed by a human virus, in mammals.
Asunto(s)
Enterovirus Humano A/inmunología , Infecciones por Enterovirus/inmunología , Inmunidad , Interferencia de ARN , ARN Viral/inmunología , Animales , Proteínas Argonautas/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Enterovirus Humano A/genética , Células HEK293 , Humanos , Mamíferos , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Mutación/genética , Ribonucleasa III/metabolismo , Proteínas Virales/inmunologíaRESUMEN
RIG-I is a key innate immune pattern-recognition receptor that triggers interferon expression upon detection of intracellular 5'triphosphate double-stranded RNA (5'ppp-dsRNA) of viral origin. RIG-I comprises N-terminal caspase activation and recruitment domains (CARDs), a DECH helicase, and a C-terminal domain (CTD). We present crystal structures of the ligand-free, autorepressed, and RNA-bound, activated states of RIG-I. Inactive RIG-I has an open conformation with the CARDs sequestered by a helical domain inserted between the two helicase moieties. ATP and dsRNA binding induce a major rearrangement to a closed conformation in which the helicase and CTD bind the blunt end 5'ppp-dsRNA with perfect complementarity but incompatibly with continued CARD binding. We propose that after initial binding of 5'ppp-dsRNA to the flexibly linked CTD, co-operative tight binding of ATP and RNA to the helicase domain liberates the CARDs for downstream signaling. These findings significantly advance our molecular understanding of the activation of innate immune signaling helicases.
Asunto(s)
Patos/metabolismo , ARN Bicatenario/metabolismo , ARN Viral/metabolismo , Receptores de Reconocimiento de Patrones/química , Receptores de Ácido Retinoico/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Pollos/inmunología , Patos/inmunología , Humanos , Modelos Moleculares , Estructura Terciaria de Proteína , ARN Bicatenario/inmunología , ARN Viral/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/inmunologíaRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently creating a global health emergency. This crisis is driving a worldwide effort to develop effective vaccines, prophylactics, and therapeutics. Nucleic acid (NA)-based treatments hold great potential to combat outbreaks of coronaviruses (CoVs) due to their rapid development, high target specificity, and the capacity to increase druggability. Here, we review key anti-CoV NA-based technologies, including antisense oligonucleotides (ASOs), siRNAs, RNA-targeting clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas), and mRNA vaccines, and discuss improved delivery methods and combination therapies with other antiviral drugs.
Asunto(s)
Vacunas contra la COVID-19 , Sistemas CRISPR-Cas , ARN Mensajero , ARN Viral , SARS-CoV-2 , COVID-19/genética , COVID-19/inmunología , COVID-19/metabolismo , COVID-19/terapia , Vacunas contra la COVID-19/genética , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/uso terapéutico , Humanos , ARN Mensajero/genética , ARN Mensajero/inmunología , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/inmunología , ARN Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/inmunología , SARS-CoV-2/metabolismoRESUMEN
SARS-CoV-2 infection causes broad-spectrum immunopathological disease, exacerbated by inflammatory co-morbidities. A better understanding of mechanisms underpinning virus-associated inflammation is required to develop effective therapeutics. Here, we discover that SARS-CoV-2 replicates rapidly in lung epithelial cells despite triggering a robust innate immune response through the activation of cytoplasmic RNA sensors RIG-I and MDA5. The inflammatory mediators produced during epithelial cell infection can stimulate primary human macrophages to enhance cytokine production and drive cellular activation. Critically, this can be limited by abrogating RNA sensing or by inhibiting downstream signalling pathways. SARS-CoV-2 further exacerbates the local inflammatory environment when macrophages or epithelial cells are primed with exogenous inflammatory stimuli. We propose that RNA sensing of SARS-CoV-2 in lung epithelium is a key driver of inflammation, the extent of which is influenced by the inflammatory state of the local environment, and that specific inhibition of innate immune pathways may beneficially mitigate inflammation-associated COVID-19.
Asunto(s)
COVID-19/inmunología , Proteína 58 DEAD Box/inmunología , Células Epiteliales/inmunología , Helicasa Inducida por Interferón IFIH1/inmunología , Macrófagos/inmunología , ARN Viral/inmunología , Receptores Inmunológicos/inmunología , SARS-CoV-2 , COVID-19/genética , COVID-19/virología , Línea Celular , Citocinas/genética , Citocinas/inmunología , Células Epiteliales/virología , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Inflamación/genética , Inflamación/inmunología , Inflamación/virología , Quinasas Janus/inmunología , Pulmón/citología , Pulmón/inmunología , Pulmón/virología , Activación de Macrófagos , FN-kappa B/inmunología , Mucosa Respiratoria/citología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/virología , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Factores de Transcripción STAT/inmunología , Replicación ViralRESUMEN
RIG-I is a key mediator of antiviral immunity, able to couple detection of infection by RNA viruses to the induction of interferons. Natural RIG-I stimulatory RNAs have variously been proposed to correspond to virus genomes, virus replication intermediates, viral transcripts, or self-RNA cleaved by RNase L. However, the relative contribution of each of these RNA species to RIG-I activation and interferon induction in virus-infected cells is not known. Here, we use three approaches to identify physiological RIG-I agonists in cells infected with influenza A virus or Sendai virus. We show that RIG-I agonists are exclusively generated by the process of virus replication and correspond to full-length virus genomes. Therefore, nongenomic viral transcripts, short replication intermediates, and cleaved self-RNA do not contribute substantially to interferon induction in cells infected with these negative strand RNA viruses. Rather, single-stranded RNA viral genomes bearing 5'-triphosphates constitute the natural RIG-I agonists that trigger cell-intrinsic innate immune responses during infection.
Asunto(s)
ARN Helicasas DEAD-box/inmunología , Proteínas de la Membrana/inmunología , Proteínas del Tejido Nervioso/inmunología , Infecciones por Virus ARN/inmunología , ARN Viral/inmunología , Animales , Línea Celular , Proteína 58 DEAD Box , Perros , Humanos , Interferones/inmunología , Ratones , Virus ARN/fisiología , Receptores de Superficie Celular , Receptores Inmunológicos , Replicación ViralRESUMEN
RIG-I detects invading viral RNA and activates the transcription factors NF-kappaB and IRF3 through the mitochondrial protein MAVS. Here we show that RNA bearing 5'-triphosphate strongly activates the RIG-I-IRF3 signaling cascade in a reconstituted system composed of RIG-I, mitochondria, and cytosol. Activation of RIG-I requires not only RNA but also polyubiquitin chains linked through lysine 63 (K63) of ubiquitin. RIG-I binds specifically to K63-polyubiquitin chains through its tandem CARD domains in a manner that depends on RNA and ATP. Mutations in the CARD domains that abrogate ubiquitin binding also impair RIG-I activation. Remarkably, unanchored K63-ubiquitin chains, which are not conjugated to any target protein, potently activate RIG-I. These ubiquitin chains function as an endogenous ligand of RIG-I in human cells. Our results delineate the mechanism of RIG-I activation, identify CARD domains as a ubiquitin sensor, and demonstrate that unanchored K63-polyubiquitin chains are signaling molecules in antiviral innate immunity.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Inmunidad Innata , ARN Viral/inmunología , Transducción de Señal , Enzimas Ubiquitina-Conjugadoras/metabolismo , Adenosina Trifosfato/metabolismo , Línea Celular , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/inmunología , Humanos , Quinasa I-kappa B/metabolismo , Factor 3 Regulador del Interferón/inmunología , Factor 3 Regulador del Interferón/metabolismo , Polifosfatos/metabolismo , Poliubiquitina/metabolismo , ARN Bicatenario/inmunología , Receptores Inmunológicos , Factores de Transcripción/metabolismo , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Mammalian cells respond to dsRNA in multiple manners. One key response to dsRNA is the activation of PKR, an eIF2α kinase, which triggers translational arrest and the formation of stress granules. However, the process of PKR activation in cells is not fully understood. In response to increased endogenous or exogenous dsRNA, we observed that PKR forms novel cytosolic condensates, referred to as dsRNA-induced foci (dRIFs). dRIFs contain dsRNA, form in proportion to dsRNA, and are enhanced by longer dsRNAs. dRIFs enrich several other dsRNA-binding proteins, including ADAR1, Stau1, NLRP1, and PACT. Strikingly, dRIFs correlate with and form before translation repression by PKR and localize to regions of cells where PKR activation is initiated. We hypothesize that dRIF formation is a mechanism that cells use to enhance the sensitivity of PKR activation in response to low levels of dsRNA or to overcome viral inhibitors of PKR activation.