RESUMEN

The current dogma of RNA-mediated innate immunity is that sensing of immunostimulatory RNA ligands is sufficient for the activation of intracellular sensors and induction of interferon (IFN) responses. Here, we report that actin cytoskeleton disturbance primes RIG-I-like receptor (RLR) activation. Actin cytoskeleton rearrangement induced by virus infection or commonly used reagents to intracellularly deliver RNA triggers the relocalization of PPP1R12C, a regulatory subunit of the protein phosphatase-1 (PP1), from filamentous actin to cytoplasmic RLRs. This allows dephosphorylation-mediated RLR priming and, together with the RNA agonist, induces effective RLR downstream signaling. Genetic ablation of PPP1R12C impairs antiviral responses and enhances susceptibility to infection with several RNA viruses including SARS-CoV-2, influenza virus, picornavirus, and vesicular stomatitis virus. Our work identifies actin cytoskeleton disturbance as a priming signal for RLR-mediated innate immunity, which may open avenues for antiviral or adjuvant design.

Asunto(s)

Actinas , COVID-19 , Citoesqueleto de Actina , Antivirales , Humanos , Interferones , Ligandos , Proteína Fosfatasa 1 , ARN , ARN Helicasas , Receptores de Ácido Retinoico/metabolismo , SARS-CoV-2

RESUMEN

Nucleic acids are used in many therapeutic modalities, including gene therapy, but their ability to trigger host immune responses in vivo can lead to decreased safety and efficacy. In the case of adeno-associated viral (AAV) vectors, studies have shown that the genome of the vector activates Toll-like receptor 9 (TLR9), a pattern recognition receptor that senses foreign DNA. Here, we engineered AAV vectors to be intrinsically less immunogenic by incorporating short DNA oligonucleotides that antagonize TLR9 activation directly into the vector genome. The engineered vectors elicited markedly reduced innate immune and T cell responses and enhanced gene expression in clinically relevant mouse and pig models across different tissues, including liver, muscle, and retina. Subretinal administration of higher-dose AAV in pigs resulted in photoreceptor pathology with microglia and T cell infiltration. These adverse findings were avoided in the contralateral eyes of the same animals that were injected with the engineered vectors. However, intravitreal injection of higher-dose AAV in macaques, a more immunogenic route of administration, showed that the engineered vector delayed but did not prevent clinical uveitis, suggesting that other immune factors in addition to TLR9 may contribute to intraocular inflammation in this model. Our results demonstrate that linking specific immunomodulatory noncoding sequences to much longer therapeutic nucleic acids can "cloak" the vector from inducing unwanted immune responses in multiple, but not all, models. This "coupled immunomodulation" strategy may widen the therapeutic window for AAV therapies as well as other DNA-based gene transfer methods.

Asunto(s)

Dependovirus , Vectores Genéticos , Animales , Dependovirus/genética , Técnicas de Transferencia de Gen , Terapia Genética , Inmunidad Innata , Ratones , Porcinos

RESUMEN

The retinoic acid-inducible gene-I (RIG-I) receptor recognizes short 5'-di- and triphosphate base-paired viral RNA and is a critical mediator of the innate immune response against viruses such as influenza A, Ebola, HIV and hepatitis C. This response is reported to require an orchestrated interaction with the tripartite motif 25 (TRIM25) B30.2 protein-interaction domain. Here, we present a novel second RIG-I-binding interface on the TRIM25 B30.2 domain that interacts with CARD1 and CARD2 (caspase activation and recruitment domains) of RIG-I and is revealed by the removal of an N-terminal α-helix that mimics dimerization of the full-length protein. Further characterization of the TRIM25 coiled-coil and B30.2 regions indicated that the B30.2 domains move freely on a flexible tether, facilitating RIG-I CARD recruitment. The identification of a dual binding mode for the TRIM25 B30.2 domain is a first for the SPRY/B30.2 domain family and may be a feature of other SPRY/B30.2 family members.

Asunto(s)

Dominio B30.2-SPRY/genética , Dominio de Reclutamiento y Activación de Caspasas/genética , Proteína 58 DEAD Box/química , Receptores Citoplasmáticos y Nucleares/química , Proteínas Recombinantes de Fusión/química , Eliminación de Secuencia , Secuencia de Aminoácidos , Animales , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Células HEK293 , Histidina/genética , Histidina/metabolismo , Humanos , Ratones , Modelos Moleculares , Oligopéptidos/genética , Oligopéptidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Inmunológicos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

Ebola virus (EBOV) infection is characterized by sporadic outbreaks caused by zoonotic transmission. Fixed changes in amino acid sequence, such as A82V in the EBOV glycoprotein (GP) that occurred early in the 2013-16 epidemic, are suspected to confer a selective advantage to the virus. We used biochemical assays of GP function to show that A82V, as well as a polymorphism in residue 544 identified in other outbreaks, enhances infection by decreasing the threshold for activation of membrane fusion activity triggered by the host factors cathepsin B and Niemann-Pick C1. Importantly, the increase in infectivity comes with the cost of decreased virus stability. Thus, emergence of a virus GP with altered properties that can affect transmission and virulence may have contributed to the severity and scope of the 2013-16 EBOV epidemic.

Asunto(s)

Sustitución de Aminoácidos , Ebolavirus/patogenicidad , Epidemias , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/virología , Proteínas Mutantes/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas Portadoras/metabolismo , Catepsina B/metabolismo , Ebolavirus/genética , Ebolavirus/fisiología , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/metabolismo , Proteínas Mutantes/genética , Proteína Niemann-Pick C1 , Selección Genética , Proteínas del Envoltorio Viral/genética , Virulencia , Internalización del Virus

RESUMEN

The cytosolic sensor MDA5 is crucial for antiviral innate immune defense against various RNA viruses including measles virus; as such, many viruses have evolved strategies to antagonize the antiviral activity of MDA5. Here, we show that measles virus escapes MDA5 detection by targeting the phosphatases PP1α and PP1γ, which regulate MDA5 activity by removing an inhibitory phosphorylation mark. The V proteins of measles virus and the related paramyxovirus Nipah virus interact with PP1α/γ, preventing PP1-mediated dephosphorylation of MDA5 and thereby its activation. The PP1 interaction with the measles V protein is mediated by a conserved PP1-binding motif in the C-terminal region of the V protein. A recombinant measles virus expressing a mutant V protein deficient in PP1 binding is unable to antagonize MDA5 and is growth impaired due to its inability to suppress interferon induction. This identifies PP1 antagonism as a mechanism employed by paramyxoviruses for evading innate immune recognition.

Asunto(s)

ARN Helicasas DEAD-box/metabolismo , Interacciones Huésped-Patógeno , Evasión Inmune , Virus del Sarampión/inmunología , Virus del Sarampión/fisiología , Fosfoproteínas/metabolismo , Proteína Fosfatasa 1/antagonistas & inhibidores , Proteínas Virales/metabolismo , Línea Celular , Humanos , Helicasa Inducida por Interferón IFIH1 , Virus Nipah/inmunología , Virus Nipah/fisiología , Proteínas Estructurales Virales/metabolismo

RESUMEN

Ubiquitylation is an important mechanism for regulating innate immune responses to viral infections. Attachment of lysine 63 (Lys(63))-linked ubiquitin chains to the RNA sensor retinoic acid-inducible gene-I (RIG-I) by the ubiquitin E3 ligase tripartite motif protein 25 (TRIM25) leads to the activation of RIG-I and stimulates production of the antiviral cytokines interferon-α (IFN-α) and IFN-ß. Conversely, Lys(48)-linked ubiquitylation of TRIM25 by the linear ubiquitin assembly complex (LUBAC) stimulates the proteasomal degradation of TRIM25, thereby inhibiting the RIG-I signaling pathway. Here, we report that ubiquitin-specific protease 15 (USP15) deubiquitylates TRIM25, preventing the LUBAC-dependent degradation of TRIM25. Through protein purification and mass spectrometry analysis, we identified USP15 as an interaction partner of TRIM25 in human cells. Knockdown of endogenous USP15 by specific small interfering RNA markedly enhanced the ubiquitylation of TRIM25. In contrast, expression of wild-type USP15, but not its catalytically inactive mutant, reduced the Lys(48)-linked ubiquitylation of TRIM25, leading to its stabilization. Furthermore, ectopic expression of USP15 enhanced the TRIM25- and RIG-I-dependent production of type I IFN and suppressed RNA virus replication. In contrast, depletion of USP15 resulted in decreased IFN production and markedly enhanced viral replication. Together, these data identify USP15 as a critical regulator of the TRIM25- and RIG-I-mediated antiviral immune response, thereby highlighting the intricate regulation of innate immune signaling.

Asunto(s)

ARN Helicasas DEAD-box/inmunología , Transducción de Señal/inmunología , Factores de Transcripción/inmunología , Ubiquitina-Proteína Ligasas/inmunología , Proteasas Ubiquitina-Específicas/inmunología , Antivirales/inmunología , Antivirales/metabolismo , Western Blotting , Células Cultivadas , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Humanos , Inmunidad Innata/inmunología , Interferón-alfa/inmunología , Interferón-alfa/metabolismo , Interferón beta/inmunología , Interferón beta/metabolismo , Lisina/metabolismo , Microscopía Confocal , Virus de la Enfermedad de Newcastle/genética , Virus de la Enfermedad de Newcastle/inmunología , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica/genética , Unión Proteica/inmunología , Proteolisis , Interferencia de ARN , Receptores Inmunológicos , Virus Sendai/inmunología , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteasas Ubiquitina-Específicas/genética , Proteasas Ubiquitina-Específicas/metabolismo , Ubiquitinación , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/inmunología , Replicación Viral/inmunología

RESUMEN

TRIM (tripartite motif) proteins primarily function as ubiquitin E3 ligases that regulate the innate immune response to infection. TRIM25 [also known as Efp (oestrogen-responsive finger protein)] has been implicated in the regulation of oestrogen receptor α signalling and in the regulation of innate immune signalling via RIG-I (retinoic acid-inducible gene-I). RIG-I senses cytosolic viral RNA and is subsequently ubiquitinated by TRIM25 at its N-terminal CARDs (caspase recruitment domains), leading to type I interferon production. The interaction with RIG-I is dependent on the TRIM25 B30.2 domain, a protein-interaction domain composed of the PRY and SPRY tandem sequence motifs. In the present study we describe the 1.8 Å crystal structure of the TRIM25 B30.2 domain, which exhibits a typical B30.2/SPRY domain fold comprising two N-terminal α-helices, thirteen ß-strands arranged into two ß-sheets and loop regions of varying lengths. A comparison with other B30.2/SPRY structures and an analysis of the loop regions identified a putative binding pocket, which is likely to be involved in binding target proteins. This was supported by mutagenesis and functional analyses, which identified two key residues (Asp(488) and Trp(621)) in the TRIM25 B30.2 domain as being critical for binding to the RIG-I CARDs.

Asunto(s)

Proteínas de Unión al ADN/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Enlace de Hidrógeno , Inmunidad Innata , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Homología Estructural de Proteína , Virosis/inmunología

RESUMEN

RIG-I and MDA5 have emerged as key cytosolic sensors for the detection of RNA viruses and lead to antiviral interferon (IFN) production. Recent studies have highlighted the importance of posttranslational modifications for controlling RIG-I antiviral activity. However, the regulation of MDA5 signal-transducing ability remains unclear. Here, we show that MDA5 signaling activity is regulated by a dynamic balance between phosphorylation and dephosphorylation of its caspase recruitment domains (CARDs). Employing a phosphatome RNAi screen, we identified PP1α and PP1γ as the primary phosphatases that are responsible for MDA5 and RIG-I dephosphorylation and that lead to their activation. Silencing of PP1α and PP1γ enhanced RIG-I and MDA5 CARD phosphorylation and reduced antiviral IFN-ß production. PP1α- and PP1γ-depleted cells were impaired in their ability to induce IFN-stimulated gene expression, which resulted in enhanced RNA virus replication. This work identifies PP1α and PP1γ as regulators of antiviral innate immune responses to various RNA viruses, including influenza virus, paramyxovirus, dengue virus, and picornavirus.

Asunto(s)

ARN Helicasas DEAD-box/inmunología , Inmunidad Innata/inmunología , Proteína Fosfatasa 1/inmunología , ARN Viral/inmunología , Animales , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Células HEK293 , Células HeLa , Humanos , Inmunidad Innata/genética , Immunoblotting , Helicasa Inducida por Interferón IFIH1 , Interferón beta/inmunología , Interferón beta/metabolismo , Ratones , Ratones Noqueados , Microscopía Confocal , Datos de Secuencia Molecular , Mutación , Fosforilación , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismo , Interferencia de ARN , ARN Viral/genética , ARN Viral/metabolismo , Receptores Inmunológicos , Transducción de Señal/genética , Transducción de Señal/inmunología , Células Vero

RESUMEN

Influenza A viruses can adapt to new host species, leading to the emergence of novel pathogenic strains. There is evidence that highly pathogenic viruses encode for non-structural 1 (NS1) proteins that are more efficient in suppressing the host immune response. The NS1 protein inhibits type-I interferon (IFN) production partly by blocking the TRIM25 ubiquitin E3 ligase-mediated Lys63-linked ubiquitination of the viral RNA sensor RIG-I, required for its optimal downstream signaling. In order to understand possible mechanisms of viral adaptation and host tropism, we examined the ability of NS1 encoded by human (Cal04), avian (HK156), swine (SwTx98) and mouse-adapted (PR8) influenza viruses to interact with TRIM25 orthologues from mammalian and avian species. Using co-immunoprecipitation assays we show that human TRIM25 binds to all tested NS1 proteins, whereas the chicken TRIM25 ortholog binds preferentially to the NS1 from the avian virus. Strikingly, none of the NS1 proteins were able to bind mouse TRIM25. Since NS1 can inhibit IFN production in mouse, we tested the impact of TRIM25 and NS1 on RIG-I ubiquitination in mouse cells. While NS1 efficiently suppressed human TRIM25-dependent ubiquitination of RIG-I 2CARD, NS1 inhibited the ubiquitination of full-length mouse RIG-I in a mouse TRIM25-independent manner. Therefore, we tested if the ubiquitin E3 ligase Riplet, which has also been shown to ubiquitinate RIG-I, interacts with NS1. We found that NS1 binds mouse Riplet and inhibits its activity to induce IFN-ß in murine cells. Furthermore, NS1 proteins of human but not swine or avian viruses were able to interact with human Riplet, thereby suppressing RIG-I ubiquitination. In conclusion, our results indicate that influenza NS1 protein targets TRIM25 and Riplet ubiquitin E3 ligases in a species-specific manner for the inhibition of RIG-I ubiquitination and antiviral IFN production.

Asunto(s)

ARN Helicasas DEAD-box/metabolismo , Virus de la Influenza A/metabolismo , Gripe Humana/metabolismo , Interferones/biosíntesis , Ubiquitinación , Proteínas no Estructurales Virales/metabolismo , Animales , Chlorocebus aethiops , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Perros , Células HeLa , Humanos , Virus de la Influenza A/genética , Gripe Humana/genética , Interferones/genética , Ratones , Ratones Noqueados , Receptores Inmunológicos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Células Vero , Proteínas no Estructurales Virales/genética