RESUMEN
The OAS-RNase L pathway is one of the oldest innate RNA sensing pathways that leads to interferon (IFN) signaling and cell death. OAS recognizes viral RNA and then activates RNase L, which subsequently cleaves both cellular and viral RNA, creating "processed RNA" as an endogenous ligand that further triggers RIG-I-like receptor signaling. However, the IFN response and antiviral activity of the OAS-RNase L pathway are weak compared to other RNA-sensing pathways. Here, we discover that the SKIV2L RNA exosome limits the antiviral capacity of the OAS-RNase L pathway. SKIV2L-deficient cells exhibit remarkably increased interferon responses to RNase L-processed RNA, resulting in heightened antiviral activity. The helicase activity of SKIV2L is indispensable for this function, acting downstream of RNase L. SKIV2L depletion increases the antiviral capacity of OAS-RNase L against RNA virus infection. Furthermore, SKIV2L loss exacerbates autoinflammation caused by human OAS1 gain-of-function mutations. Taken together, our results identify SKIV2L as a critical barrier to OAS-RNase L-mediated antiviral immunity that could be therapeutically targeted to enhance the activity of a basic antiviral pathway.
RESUMEN
High expression of 2',5'-oligoadenylate synthetase 1 (OAS1), which adds AMP residues in 2',5' linkage to a variety of substrates, is observed in many cancers as a part of the interferon-related DNA damage resistance signature (IRDS). Poly(ADP-ribose) (PAR) is rapidly synthesized from NAD+ at sites of DNA damage to facilitate repair, but excessive PAR synthesis due to extensive DNA damage results in cell death by energy depletion and/or activation of PAR-dependent programmed cell death pathways. We find that OAS1 adds AMP residues in 2',5' linkage to PAR, inhibiting its synthesis in vitro and reducing its accumulation in cells. Increased OAS1 expression substantially improves cell viability following DNA-damaging treatments that stimulate PAR synthesis during DNA repair. We conclude that high expression of OAS1 in cancer cells promotes their ability to survive DNA damage by attenuating PAR synthesis and thus preventing cell death.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/biosíntesis , Daño del ADN , Regulación Enzimológica de la Expresión Génica , Poli ADP Ribosilación , 2',5'-Oligoadenilato Sintetasa/genética , Adenosina Monofosfato/genética , Adenosina Monofosfato/metabolismo , Muerte Celular , Línea Celular Transformada , HumanosRESUMEN
Infection with the flavivirus Zika virus (ZIKV) can result in tissue tropism, disease outcome, and route of transmission distinct from those of other flaviviruses; therefore, we aimed to identify host machinery that exclusively promotes the ZIKV replication cycle, which can inform on differences at the organismal level. We previously reported that deletion of the host antiviral ribonuclease L (RNase L) protein decreases ZIKV production. Canonical RNase L catalytic activity typically restricts viral infection, including that of the flavivirus dengue virus (DENV), suggesting an unconventional, proviral RNase L function during ZIKV infection. In this study, we reveal that an inactive form of RNase L supports assembly of ZIKV replication factories (RFs) to enhance infectious virus production. Compared with the densely concentrated ZIKV RFs generated with RNase L present, deletion of RNase L induced broader subcellular distribution of ZIKV replication intermediate double-stranded RNA (dsRNA) and NS3 protease, two constituents of ZIKV RFs. An inactive form of RNase L was sufficient to contain ZIKV genome and dsRNA within a smaller RF area, which subsequently increased infectious ZIKV release from the cell. Inactive RNase L can interact with cytoskeleton, and flaviviruses remodel cytoskeleton to construct RFs. Thus, we used the microtubule-stabilization drug paclitaxel to demonstrate that ZIKV repurposes RNase L to facilitate the cytoskeleton rearrangements required for proper generation of RFs. During infection with flaviviruses DENV or West Nile Kunjin virus, inactive RNase L did not improve virus production, suggesting that a proviral RNase L role is not a general feature of all flavivirus infections.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/metabolismo , Endorribonucleasas/metabolismo , Interacciones Huésped-Patógeno , Replicación Viral , Virus Zika/fisiología , 2',5'-Oligoadenilato Sintetasa/genética , Células A549 , Endorribonucleasas/genética , HumanosRESUMEN
Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection; induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung; and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, whereas PKR activation is evident in iAT2 and iCM. In SARS-CoV-2-infected Calu-3 and A549ACE2 lung-derived cell lines, IFN induction remains relatively weak; however, activation of OAS-RNase L and PKR is observed. This is in contrast to Middle East respiratory syndrome (MERS)-CoV, which effectively inhibits IFN signaling and OAS-RNase L and PKR pathways, but is similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, OAS-RNase L and PKR are activated in MAVS knockout A549ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2.
Asunto(s)
Células Epiteliales/inmunología , Células Epiteliales/virología , Inmunidad Innata , Pulmón/patología , Miocitos Cardíacos/inmunología , Miocitos Cardíacos/virología , ARN Bicatenario/metabolismo , SARS-CoV-2/inmunología , Células A549 , Endorribonucleasas/metabolismo , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Coronavirus del Síndrome Respiratorio de Oriente Medio/fisiología , Nariz/virología , Replicación Viral , eIF-2 QuinasaRESUMEN
The oligoadenylate synthetase (OAS)-RNase L system is an IFN-inducible antiviral pathway activated by viral infection. Viral double-stranded (ds) RNA activates OAS isoforms that synthesize the second messenger 2-5A, which binds and activates the pseudokinase-endoribonuclease RNase L. In cells, OAS activation is tamped down by ADAR1, an adenosine deaminase that destabilizes dsRNA. Mutation of ADAR1 is one cause of Aicardi-Goutières syndrome (AGS), an interferonopathy in children. ADAR1 deficiency in human cells can lead to RNase L activation and subsequent cell death. To evaluate RNase L as a possible therapeutic target for AGS, we sought to identify small-molecule inhibitors of RNase L. A 500-compound library of protein kinase inhibitors was screened for modulators of RNase L activity in vitro. We identified ellagic acid (EA) as a hit with 10-fold higher selectivity against RNase L compared with its nearest paralog, IRE1. SAR analysis identified valoneic acid dilactone (VAL) as a superior inhibitor of RNase L, with 100-fold selectivity over IRE1. Mechanism-of-action analysis indicated that EA and VAL do not bind to the pseudokinase domain of RNase L despite acting as ATP competitive inhibitors of the protein kinase CK2. VAL is nontoxic and functional in cells, although with a 1,000-fold decrease in potency, as measured by RNA cleavage activity in response to treatment with dsRNA activator or by rescue of cell lethality resulting from self dsRNA induced by ADAR1 deficiency. These studies lay the foundation for understanding novel modes of regulating RNase L function using small-molecule inhibitors and avenues of therapeutic potential.
Asunto(s)
Adenosina Desaminasa/deficiencia , Enfermedades Autoinmunes del Sistema Nervioso/enzimología , Endorribonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Malformaciones del Sistema Nervioso/enzimología , Fenol/farmacología , 2',5'-Oligoadenilato Sintetasa/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , Nucleótidos de Adenina/metabolismo , Adenosina Desaminasa/genética , Enfermedades Autoinmunes del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/fisiopatología , Muerte Celular/efectos de los fármacos , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Inhibidores Enzimáticos/química , Humanos , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/fisiopatología , Oligorribonucleótidos/metabolismo , Fenol/química , Proteínas de Unión al ARN/genéticaRESUMEN
RNase L is an ankyrin repeat domain-containing dual endoribonuclease-pseudokinase that is activated by unusual 2,'5'-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferon-regulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here we present a functional characterization of 2.5 Å and 3.25 Å X-ray crystal and small-angle X-ray scattering structures of RNase L bound to a natural 2-5A activator with and without ADP or the nonhydrolysable ATP mimetic AMP-PNP. These studies reveal how recognition of 2-5A through interactions with the ankyrin repeat domain and the pseudokinase domain, together with nucleotide binding, imposes a rigid intertwined dimer configuration that is essential for RNase catalytic and antiviral functions. The involvement of the pseudokinase domain of RNase L in 2-5A sensing, nucleotide binding, dimerization, and ribonuclease functions highlights the evolutionary adaptability of the eukaryotic protein kinase fold.
Asunto(s)
Nucleótidos de Adenina/química , Endorribonucleasas/química , Oligorribonucleótidos/química , Adenosina Difosfato/química , Adenilil Imidodifosfato/química , Animales , Repetición de Anquirina , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Virus de la Encefalomiocarditis , Endorribonucleasas/genética , Endorribonucleasas/fisiología , Células HeLa , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Picornaviridae , Estructura Terciaria de Proteína , Dispersión de Radiación , Relación Estructura-Actividad , Sus scrofaRESUMEN
Drugs that reverse epigenetic silencing, such as the DNA methyltransferase inhibitor (DNMTi) 5-azacytidine (AZA), have profound effects on transcription and tumor cell survival. AZA is an approved drug for myelodysplastic syndromes and acute myeloid leukemia, and is under investigation for different solid malignant tumors. AZA treatment generates self, double-stranded RNA (dsRNA), transcribed from hypomethylated repetitive elements. Self dsRNA accumulation in DNMTi-treated cells leads to type I IFN production and IFN-stimulated gene expression. Here we report that cell death in response to AZA treatment occurs through the 2',5'-oligoadenylate synthetase (OAS)-RNase L pathway. OASs are IFN-induced enzymes that synthesize the RNase L activator 2-5A in response to dsRNA. Cells deficient in RNase L or OAS1 to 3 are highly resistant to AZA, as are wild-type cells treated with a small-molecule inhibitor of RNase L. A small-molecule inhibitor of c-Jun NH2-terminal kinases (JNKs) also antagonizes RNase L-dependent cell death in response to AZA, consistent with a role for JNK in RNase L-induced apoptosis. In contrast, the rates of AZA-induced and RNase L-dependent cell death were increased by transfection of 2-5A, by deficiencies in ADAR1 (which edits and destabilizes dsRNA), PDE12 or AKAP7 (which degrade 2-5A), or by ionizing radiation (which induces IFN-dependent signaling). Finally, OAS1 expression correlates with AZA sensitivity in the NCI-60 set of tumor cell lines, suggesting that the level of OAS1 can be a biomarker for predicting AZA sensitivity of tumor cells. These studies may eventually lead to pharmacologic strategies for regulating the antitumor activity and toxicity of AZA and related drugs.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/metabolismo , Azacitidina/farmacología , Desmetilación del ADN , Endorribonucleasas/metabolismo , Inmunidad Innata , Células A549 , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Muerte Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Isoenzimas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Radiación Ionizante , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The 2',5'-oligoadenylate (2-5A) synthetase (OAS)-RNase L system is an IFN-induced antiviral pathway. RNase L activity depends on 2-5A, synthesized by OAS. Although all three enzymatically active OAS proteins in humans--OAS1, OAS2, and OAS3--synthesize 2-5A upon binding dsRNA, it is unclear which are responsible for RNase L activation during viral infection. We used clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) technology to engineer human A549-derived cell lines in which each of the OAS genes or RNase L is knocked out. Upon transfection with poly(rI):poly(rC), a synthetic surrogate for viral dsRNA, or infection with each of four viruses from different groups (West Nile virus, Sindbis virus, influenza virus, or vaccinia virus), OAS1-KO and OAS2-KO cells synthesized amounts of 2-5A similar to those synthesized in parental wild-type cells, causing RNase L activation as assessed by rRNA degradation. In contrast, OAS3-KO cells synthesized minimal 2-5A, and rRNA remained intact, similar to infected RNase L-KO cells. All four viruses replicated to higher titers in OAS3-KO or RNase L-KO A549 cells than in parental, OAS1-KO, or OAS2-KO cells, demonstrating the antiviral effects of OAS3. OAS3 displayed a higher affinity for dsRNA in intact cells than either OAS1 or OAS2, consistent with its dominant role in RNase L activation. Finally, the requirement for OAS3 as the major OAS isoform responsible for RNase L activation was not restricted to A549 cells, because OAS3-KO cells derived from two other human cell lines also were deficient in RNase L activation.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/metabolismo , Endorribonucleasas/metabolismo , Virosis/metabolismo , 2',5'-Oligoadenilato Sintetasa/antagonistas & inhibidores , 2',5'-Oligoadenilato Sintetasa/genética , Infecciones por Alphavirus/genética , Infecciones por Alphavirus/metabolismo , Sistemas CRISPR-Cas , Línea Celular , Endorribonucleasas/antagonistas & inhibidores , Endorribonucleasas/genética , Activación Enzimática , Técnicas de Inactivación de Genes , Humanos , Gripe Humana/genética , Gripe Humana/metabolismo , Modelos Biológicos , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Virus Sindbis , Vaccinia/genética , Vaccinia/metabolismo , Virosis/genética , Fiebre del Nilo Occidental/genética , Fiebre del Nilo Occidental/metabolismoRESUMEN
Retrotransposons are mobile genetic elements, and their mobility can lead to genomic instability. Retrotransposon insertions are associated with a diverse range of sporadic diseases, including cancer. Thus, it is not a surprise that multiple host defense mechanisms suppress retrotransposition. The 2',5'-oligoadenylate (2-5A) synthetase (OAS)-RNase L system is a mechanism for restricting viral infections during the interferon antiviral response. Here, we investigated a potential role for the OAS-RNase L system in the restriction of retrotransposons. Expression of wild type (WT) and a constitutively active form of RNase L (NΔ385), but not a catalytically inactive RNase L mutant (R667A), impaired the mobility of engineered human LINE-1 (L1) and mouse intracisternal A-type particle retrotransposons in cultured human cells. Furthermore, WT RNase L, but not an inactive RNase L mutant (R667A), reduced L1 RNA levels and subsequent expression of the L1-encoded proteins (ORF1p and ORF2p). Consistently, confocal immunofluorescent microscopy demonstrated that WT RNase L, but not RNase L R667A, prevented formation of L1 cytoplasmic foci. Finally, siRNA-mediated depletion of endogenous RNase L in a human ovarian cancer cell line (Hey1b) increased the levels of L1 retrotransposition by â¼2-fold. Together, these data suggest that RNase L might function as a suppressor of structurally distinct retrotransposons.
Asunto(s)
Endorribonucleasas/metabolismo , Genes de Partícula A Intracisternal , Elementos de Nucleótido Esparcido Largo , Animales , Línea Celular Tumoral , Células HeLa , Humanos , Ratones , ARN Mensajero/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Efficient and productive virus infection often requires viral countermeasures that block innate immunity. The IFN-inducible 2',5'-oligoadenylate (2-5A) synthetases (OASs) and ribonuclease (RNase) L are components of a potent host antiviral pathway. We previously showed that murine coronavirus (MHV) accessory protein ns2, a 2H phosphoesterase superfamily member, is a phosphodiesterase (PDE) that cleaves 2-5A, thereby preventing activation of RNase L. The PDE activity of ns2 is required for MHV replication in macrophages and for hepatitis. Here, we show that group A rotavirus (RVA), an important cause of acute gastroenteritis in children worldwide, encodes a similar PDE. The RVA PDE forms the carboxy-terminal domain of the minor core protein VP3 (VP3-CTD) and shares sequence and predicted structural homology with ns2, including two catalytic HxT/S motifs. Bacterially expressed VP3-CTD exhibited 2',5'-PDE activity, which cleaved 2-5A in vitro. In addition, VP3-CTD expressed transiently in mammalian cells depleted 2-5A levels induced by OAS activation with poly(rI):poly(rC), preventing RNase L activation. In the context of recombinant chimeric MHV expressing inactive ns2, VP3-CTD restored the ability of the virus to replicate efficiently in macrophages or in the livers of infected mice, whereas mutant viruses expressing inactive VP3-CTD (H718A or H798R) were attenuated. In addition, chimeric viruses expressing either active ns2 or VP3-CTD, but not nonfunctional equivalents, were able to protect ribosomal RNA from RNase L-mediated degradation. Thus, VP3-CTD is a 2',5'-PDE able to functionally substitute for ns2 in MHV infection. Remarkably, therefore, two disparate RNA viruses encode proteins with homologous 2',5'-PDEs that antagonize activation of innate immunity.
Asunto(s)
Inmunidad Innata/inmunología , Hidrolasas Diéster Fosfóricas/inmunología , Virus ARN/inmunología , Proteínas no Estructurales Virales/inmunología , 2',5'-Oligoadenilato Sintetasa/inmunología , 2',5'-Oligoadenilato Sintetasa/metabolismo , Nucleótidos de Adenina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Sitios de Unión/inmunología , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/metabolismo , Línea Celular , Células Cultivadas , Endorribonucleasas/genética , Endorribonucleasas/inmunología , Endorribonucleasas/metabolismo , Interacciones Huésped-Patógeno/inmunología , Humanos , Immunoblotting , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/virología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Virus de la Hepatitis Murina/inmunología , Virus de la Hepatitis Murina/metabolismo , Virus de la Hepatitis Murina/fisiología , Mutación , Oligorribonucleótidos/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/virología , Virus ARN/metabolismo , Virus ARN/fisiología , Rotavirus/inmunología , Rotavirus/metabolismo , Rotavirus/fisiología , Homología de Secuencia de Aminoácido , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
The use of lytic viruses to preferentially infect and eliminate cancer cells while sparing normal cells is a promising experimental therapeutic approach for treating cancer. However, the efficacy of oncolytic virotherapy is often limited by two innate immunity pathways, the protein kinase PKR and the 2'-5'-oligoadenylate (OAS)/RNase L systems, which are widely present in many but not all tumor cell types. Previously, we reported that the anticancer drug, sunitinib, an inhibitor of VEGF-R and PDGF-R, has off-target effects against both PKR and RNase L. Here we show that combining sunitinib treatments with infection by an oncolytic virus, vesicular stomatitis virus (VSV), led to the elimination of prostate, breast, and kidney malignant tumors in mice. In contrast, either virus or sunitinib alone slowed tumor progression but did not eliminate tumors. In prostate tumors excised from treated mice, sunitinib decreased levels of the phosphorylated form of translation initiation factor, eIF2-α, a substrate of PKR, by 10-fold while increasing median viral titers by 23-fold. The sunitinib/VSV regimen caused complete and sustained tumor regression in both immunodeficient and immunocompetent animals. Results indicate that transient inhibition of innate immunity with sunitinib enhances oncolytic virotherapy allowing the recovery of tumor-bearing animals.
Asunto(s)
Antineoplásicos/farmacología , Inmunidad Innata/efectos de los fármacos , Indoles/farmacología , Viroterapia Oncolítica , Virus Oncolíticos/fisiología , Pirroles/farmacología , Vesiculovirus/fisiología , Animales , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/terapia , Línea Celular Tumoral , Terapia Combinada , Endorribonucleasas/antagonistas & inhibidores , Endorribonucleasas/metabolismo , Femenino , Indoles/administración & dosificación , Neoplasias Renales/patología , Neoplasias Renales/terapia , Masculino , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Virus Oncolíticos/inmunología , Virus Oncolíticos/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapia , Pirroles/administración & dosificación , Sunitinib , Vesiculovirus/genética , eIF-2 Quinasa/antagonistas & inhibidores , eIF-2 Quinasa/metabolismoRESUMEN
Antiviral innate immunity is initiated in response to RNA molecules that are produced in virus-infected cells. These RNAs activate signalling cascades that activate the genes that encode alpha- and beta-interferon (IFN). Signalling occurs through the interaction of the RNAs with either of two pathogen recognition receptors, retinoic acid-inducible gene-I (RIG-I, also known as DDX58) and melanoma differentiation associated gene-5 (MDA5, also known as IFIH1), which contain amino-terminal caspase activation and recruitment domains (CARD) and carboxy-terminal DExD/H Box RNA helicase motifs. RIG-I and MDA5 interact with another CARD protein, interferon-beta promotor stimulator protein-1 (IPS-1, also known as MAVS, VISA and Cardif), in the mitochondrial membrane, which relays the signal through the transcription factors interferon regulatory factor 3 (IRF-3) and nuclear factor (NF)-kappaB to the IFN-beta gene. Although the signalling pathway is well understood, the origin of the RNA molecules that initiate these processes is not. Here we show that activation of the antiviral endoribonuclease, RNase L, by 2',5'-linked oligoadenylate (2-5A) produces small RNA cleavage products from self-RNA that initiate IFN production. Accordingly, mouse embryonic fibroblasts lacking RNase L were resistant to the induction of IFN-beta expression in response to 2-5A, dsRNA or viral infection. Single-stranded regions of RNA are cleaved 3' of UpUp and UpAp sequences by RNase L during viral infections, resulting in small, often duplex, RNAs. We show that small self-RNAs produced by the action of RNase L on cellular RNA induce IFN-beta expression and that the signalling involves RIG-I, MDA5 and IPS-1. Mice lacking RNase L produce significantly less IFN-beta during viral infections than infected wild-type mice. Furthermore, activation of RNase L with 2-5A in vivo induced the expression of IFN-beta in wild-type but not RNase L-deficient mice. Our results indicate that RNase L has an essential role in the innate antiviral immune response that relieves the requirement for direct sensing of non-self RNA.
Asunto(s)
Infecciones por Cardiovirus/inmunología , Virus de la Encefalomiocarditis/inmunología , Endorribonucleasas/metabolismo , Inmunidad Innata/inmunología , ARN/inmunología , ARN/metabolismo , Nucleótidos de Adenina/inmunología , Animales , Infecciones por Cardiovirus/virología , Virus de la Encefalomiocarditis/fisiología , Endorribonucleasas/deficiencia , Endorribonucleasas/genética , Fibroblastos , Interferón beta/biosíntesis , Interferón beta/genética , Interferón beta/inmunología , Ratones , Oligorribonucleótidos/inmunología , Regiones Promotoras Genéticas/genética , ARN/química , ARN Bicatenario/inmunología , Transducción de Señal , Activación Transcripcional/genéticaRESUMEN
Multisystem inflammatory syndrome in children (MIS-C) is a rare and severe condition that follows benign COVID-19. We report autosomal recessive deficiencies of OAS1, OAS2, or RNASEL in five unrelated children with MIS-C. The cytosolic double-stranded RNA (dsRNA)-sensing OAS1 and OAS2 generate 2'-5'-linked oligoadenylates (2-5A) that activate the single-stranded RNA-degrading ribonuclease L (RNase L). Monocytic cell lines and primary myeloid cells with OAS1, OAS2, or RNase L deficiencies produce excessive amounts of inflammatory cytokines upon dsRNA or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) stimulation. Exogenous 2-5A suppresses cytokine production in OAS1-deficient but not RNase L-deficient cells. Cytokine production in RNase L-deficient cells is impaired by MDA5 or RIG-I deficiency and abolished by mitochondrial antiviral-signaling protein (MAVS) deficiency. Recessive OAS-RNase L deficiencies in these patients unleash the production of SARS-CoV-2-triggered, MAVS-mediated inflammatory cytokines by mononuclear phagocytes, thereby underlying MIS-C.
Asunto(s)
COVID-19 , Citocinas , Endorribonucleasas , SARS-CoV-2 , Síndrome de Respuesta Inflamatoria Sistémica , Niño , Humanos , COVID-19/inmunología , Citocinas/genética , Citocinas/inmunología , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , ARN Bicatenario , SARS-CoV-2/genética , Síndrome de Respuesta Inflamatoria Sistémica/genéticaRESUMEN
RNase L and RNA-dependent protein kinase (PKR) are effectors of the interferon antiviral response that share homology in their pseudokinase and protein kinase domains, respectively. Sunitinib is an orally available, ATP-competitive inhibitor of VEGF and PDGF receptors used clinically to suppress angiogenesis and tumor growth. Sunitinib also impacts IRE1, an endoplasmic reticulum protein involved in the unfolded protein response that is closely related to RNase L. Here, we report that sunitinib is a potent inhibitor of both RNase L and PKR with IC(50) values of 1.4 and 0.3 µM, respectively. In addition, flavonol activators of IRE1 inhibited RNase L. Sunitinib treatment of wild type (WT) mouse embryonic fibroblasts resulted in about a 12-fold increase in encephalomyocarditis virus titers. However, sunitinib had no effect on encephalomyocarditis virus growth in cells lacking both PKR and RNase L. Furthermore, oral delivery of sunitinib in WT mice resulted in 10-fold higher viral titers in heart tissues while suppressing by about 2-fold the IFN-ß levels. In contrast, sunitinib had no effect on viral titers in mice deficient in both RNase L and PKR. Also, sunitinib reduced mean survival times from 12 to 6 days in virus-infected WT mice while having no effect on survival of mice lacking both RNase L and PKR. Results indicate that sunitinib treatments prevent antiviral innate immune responses mediated by RNase L and PKR.
Asunto(s)
Antineoplásicos/farmacología , Infecciones por Cardiovirus/inmunología , Endorribonucleasas/antagonistas & inhibidores , Inmunidad Innata/efectos de los fármacos , Indoles/farmacología , Proteínas de la Membrana/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Pirroles/farmacología , eIF-2 Quinasa/antagonistas & inhibidores , Animales , Infecciones por Cardiovirus/enzimología , Infecciones por Cardiovirus/genética , Virus de la Encefalomiocarditis , Endorribonucleasas/genética , Endorribonucleasas/inmunología , Endorribonucleasas/metabolismo , Humanos , Inmunidad Innata/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/metabolismo , Sunitinib , eIF-2 Quinasa/genética , eIF-2 Quinasa/inmunologíaRESUMEN
Xenotropic murine leukemia-related virus (XMRV) was identified in association with human prostate cancer and chronic fatigue syndrome. To examine the infection potential, kinetics, and tissue distribution of XMRV in an animal model, we inoculated five macaques with XMRV intravenously. XMRV established a persistent, chronic disseminated infection, with low transient viremia and provirus in blood lymphocytes during acute infection. Although undetectable in blood after about a month, XMRV viremia was reactivated at 9 months, confirming the chronicity of the infection. Furthermore, XMRV Gag was detected in tissues throughout, with wide dissemination throughout the period of monitoring. Surprisingly, XMRV infection showed organ-specific cell tropism, infecting CD4 T cells in lymphoid organs including the gastrointestinal lamina propria, alveolar macrophages in lung, and epithelial/interstitial cells in other organs, including the reproductive tract. Of note, in spite of the intravenous inoculation, extensive XMRV replication was noted in prostate during acute but not chronic infection even though infected cells were still detectable by fluorescence in situ hybridization (FISH) in prostate at 5 and 9 months postinfection. Marked lymphocyte activation occurred immediately postinfection, but antigen-specific cellular responses were undetectable. Antibody responses were elicited and boosted upon reexposure, but titers decreased rapidly, suggesting low antigen stimulation over time. Our findings establish a nonhuman primate model to study XMRV replication/dissemination, transmission, pathogenesis, immune responses, and potential future therapies.
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Anticuerpos Antivirales/sangre , Modelos Animales de Enfermedad , Macaca mulatta/virología , Enfermedades de los Primates/virología , Infecciones por Retroviridae/virología , Virus Relacionado con el Virus Xenotrópico de la Leucemia Murina/inmunología , Virus Relacionado con el Virus Xenotrópico de la Leucemia Murina/patogenicidad , Animales , Linfocitos T CD4-Positivos/virología , Enfermedad Crónica , Células Epiteliales/virología , Humanos , Linfocitos/virología , Macrófagos/virología , Masculino , Enfermedades de los Primates/inmunología , Enfermedades de los Primates/patología , Provirus/aislamiento & purificación , Infecciones por Retroviridae/inmunología , Infecciones por Retroviridae/patología , Tropismo Viral , Viremia , Activación Viral , Latencia del VirusRESUMEN
The pseudokinase-endoribonuclease RNase L plays important roles in antiviral innate immunity and is also implicated in many other cellular activities. The inhibition of RNase L showed therapeutic potential for Aicardi-Goutières syndrome (AGS). Thus, RNase L is a promising drug target. In this study, using an enzyme assay and NMR screening, we discovered 13 inhibitory fragments against RNase L. Cocrystal structures of RNase L separately complexed with two different fragments were determined in which both fragments bound to the ATP-binding pocket of the pseudokinase domain. Myricetin, vitexin, and hyperoside, three natural products sharing similar scaffolds with the fragment AC40357, demonstrated a potent inhibitory activity in vitro. In addition, myricetin has a promising cellular inhibitory activity. A cocrystal structure of RNase L with myricetin provided a structural basis for inhibitor design by allosterically modulating the ribonuclease activity. Our findings demonstrate that fragment screening can lead to the discovery of natural product inhibitors of RNase L.
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Productos Biológicos/farmacología , Descubrimiento de Drogas , Endorribonucleasas/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , HumanosRESUMEN
Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus originally identified in a subset of prostate cancer patients. Because androgens stimulate prostate tumors and some retroviruses, we investigated the effects of dihydrotestosterone (DHT) on XMRV transcription and replication. Transcription from the XMRV U3 region was stimulated up to 2-fold by DHT, but only in cells containing a functional androgen receptor. Mutations in the glucocorticoid response element (GRE) of XMRV impaired basal transcription and androgen responsiveness. Furthermore, DHT stimulated XMRV replication 3-fold, whereas androgen inhibitors (casodex and flutamide) suppressed viral growth up to 3-fold. Findings suggest that integration of the XMRV long terminal repeat (LTR) into host DNA could impart androgen stimulation on cellular genes.
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Dihidrotestosterona/farmacología , Virus de la Leucemia Murina/fisiología , Transcripción Genética/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Línea Celular , Humanos , Virus de la Leucemia Murina/genéticaRESUMEN
The 2',5'-oligoadenylate (2-5A)-dependent endoribonuclease, RNase L, is a principal mediator of the interferon (IFN) antiviral response. Therefore, the regulation of cellular levels of 2-5A is a key point of control in antiviral innate immunity. Cellular 2-5A levels are determined by IFN-inducible 2',5'-oligoadenylate synthetases (OASs) and by enzymes that degrade 2-5A. Importantly, many coronaviruses (CoVs) and rotaviruses encode 2-5A-degrading enzymes, thereby antagonizing RNase L and its antiviral effects. A-kinase-anchoring protein 7 (AKAP7), a mammalian counterpart, could possibly limit tissue damage from excessive or prolonged RNase L activation during viral infections or from self-double-stranded RNAs that activate OAS. We show that these enzymes, members of the two-histidine phosphoesterase (2H-PE) superfamily, constitute a subfamily referred here as 2',5'-PEs. 2',5'-PEs from the mouse CoV mouse hepatitis virus (MHV) (NS2), Middle East respiratory syndrome coronavirus (MERS-CoV) (NS4b), group A rotavirus (VP3), and mouse (AKAP7) were investigated for their evolutionary relationships and activities. While there was no activity against 3',5'-oligoribonucleotides, they all cleaved 2',5'-oligoadenylates efficiently but with variable activity against other 2',5'-oligonucleotides. The 2',5'-PEs are shown to be metal ion-independent enzymes that cleave trimer 2-5A (2',5'-p3A3) producing mono- or diadenylates with 2',3'-cyclic phosphate termini. Our results suggest that the elimination of 2-5A might be the sole function of viral 2',5'-PEs, thereby promoting viral escape from innate immunity by preventing or limiting the activation of RNase L. IMPORTANCE Viruses often encode accessory proteins that antagonize the host antiviral immune response. Here, we probed the evolutionary relationships and biochemical activities of two-histidine phosphoesterases (2H-PEs) that allow some coronaviruses and rotaviruses to counteract antiviral innate immunity. In addition, we investigated the mammalian enzyme AKAP7, which has homology and shared activities with the viral enzymes and might reduce self-injury. These viral and host enzymes, which we refer to as 2',5'-PEs, specifically degrade 2',5'-oligoadenylate activators of the antiviral enzyme RNase L. We show that the host and viral enzymes are metal ion independent and exclusively cleave 2',5'- and not 3',5'-phosphodiester bonds, producing cleavage products with cyclic 2',3'-phosphate termini. Our study defines 2',5'-PEs as enzymes that share characteristic conserved features with the 2H-PE superfamily but have specific and distinct biochemical cleavage activities. These findings may eventually lead to pharmacological strategies for developing antiviral drugs against coronaviruses, rotaviruses, and other viruses.
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Proteínas de Anclaje a la Quinasa A/metabolismo , Nucleótidos de Adenina/metabolismo , Endorribonucleasas/metabolismo , Coronavirus del Síndrome Respiratorio de Oriente Medio/enzimología , Virus de la Hepatitis Murina/enzimología , Oligorribonucleótidos/metabolismo , Rotavirus/enzimología , Animales , Humanos , Inmunidad Innata/inmunología , Interferones/inmunología , RatonesRESUMEN
Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection, induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung, and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, while PKR activation is evident in iAT2 and iCM. In SARS-CoV-2 infected Calu-3 and A549 ACE2 lung-derived cell lines, IFN induction remains relatively weak; however activation of OAS-RNase L and PKR is observed. This is in contrast to MERS-CoV, which effectively inhibits IFN signaling as well as OAS-RNase L and PKR pathways, but similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, both OAS-RNase L and PKR are activated in MAVS knockout A549 ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549 ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2. SIGNIFICANCE: SARS-CoV-2 emergence in late 2019 led to the COVID-19 pandemic that has had devastating effects on human health and the economy. Early innate immune responses are essential for protection against virus invasion. While inadequate innate immune responses are associated with severe COVID-19 diseases, understanding of the interaction of SARS-CoV-2 with host antiviral pathways is minimal. We have characterized the innate immune response to SARS-CoV-2 infections in relevant respiratory tract derived cells and cardiomyocytes and found that SARS-CoV-2 activates two antiviral pathways, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR), while inducing minimal levels of interferon. This in contrast to MERS-CoV which inhibits all three pathways. Activation of these pathways may contribute to the distinctive pathogenesis of SARS-CoV-2.