RESUMO
The SARS-CoV-2 pandemic has caused extreme human suffering and economic harm. We generated and characterized a new mouse-adapted SARS-CoV-2 virus that captures multiple aspects of severe COVID-19 disease in standard laboratory mice. This SARS-CoV-2 model exhibits the spectrum of morbidity and mortality of COVID-19 disease as well as aspects of host genetics, age, cellular tropisms, elevated Th1 cytokines, and loss of surfactant expression and pulmonary function linked to pathological features of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). This model can rapidly access existing mouse resources to elucidate the role of host genetics, underlying molecular mechanisms governing SARS-CoV-2 pathogenesis, and the protective or pathogenic immune responses related to disease severity. The model promises to provide a robust platform for studies of ALI and ARDS to evaluate vaccine and antiviral drug performance, including in the most vulnerable populations (i.e., the aged) using standard laboratory mice.
Assuntos
Lesão Pulmonar Aguda/patologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Animais , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , COVID-19 , Linhagem Celular , Quimiocinas/sangue , Infecções por Coronavirus/mortalidade , Infecções por Coronavirus/virologia , Citocinas/sangue , Modelos Animais de Doenças , Feminino , Humanos , Pulmão/patologia , Pulmão/fisiologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pandemias , Pneumonia Viral/mortalidade , Pneumonia Viral/virologia , Síndrome do Desconforto Respiratório/patologia , SARS-CoV-2 , Índice de Gravidade de Doença , Taxa de SobrevidaRESUMO
NLRX1 is unique among the nucleotide-binding-domain and leucine-rich-repeat (NLR) proteins in its mitochondrial localization and ability to negatively regulate antiviral innate immunity dependent on the adaptors MAVS and STING. However, some studies have suggested a positive regulatory role for NLRX1 in inducing antiviral responses. We found that NLRX1 exerted opposing regulatory effects on viral activation of the transcription factors IRF1 and IRF3, which might potentially explain such contradictory results. Whereas NLRX1 suppressed MAVS-mediated activation of IRF3, it conversely facilitated virus-induced increases in IRF1 expression and thereby enhanced control of viral infection. NLRX1 had a minimal effect on the transcription of IRF1 mediated by the transcription factor NF-kB and regulated the abundance of IRF1 post-transcriptionally by preventing translational shutdown mediated by the double-stranded RNA (dsRNA)-activated kinase PKR and thereby allowed virus-induced increases in the abundance of IRF1 protein.
Assuntos
Hepacivirus/imunologia , Hepatite C/imunologia , Imunidade Inata/imunologia , Fator Regulador 1 de Interferon/imunologia , Fator Regulador 3 de Interferon/imunologia , Proteínas Mitocondriais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Células Cultivadas , Ativação Enzimática/imunologia , Células HEK293 , Hepatite C/virologia , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Fator Regulador 1 de Interferon/metabolismo , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , NF-kappa B/metabolismo , RNA Viral/genética , Vírus Sendai/imunologia , eIF-2 Quinase/metabolismoRESUMO
The functional significance of protein diversification through translational regulation in mammals is largely unexplored. Brubaker et al. now describe the generation of two functionally distinct mammalian proteins, MAVS and miniMAVS, from a single bicistronic mRNA and suggest that noncanonical translation may impact multiple players in innate immune regulation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Regulação da Expressão Gênica , Imunidade Inata , Biossíntese de Proteínas , Animais , HumanosRESUMO
Among the caspases that cause regulated cell death, a unique function for caspase-7 has remained elusive. Caspase-3 performs apoptosis, whereas caspase-7 is typically considered an inefficient back-up. Caspase-1 activates gasdermin D pores to lyse the cell; however, caspase-1 also activates caspase-7 for unknown reasons1. Caspases can also trigger cell-type-specific death responses; for example, caspase-1 causes the extrusion of intestinal epithelial cell (IECs) in response to infection with Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium)2,3. Here we show in both organoids and mice that caspase-7-deficient IECs do not complete extrusion. Mechanistically, caspase-7 counteracts gasdermin D pores and preserves cell integrity by cleaving and activating acid sphingomyelinase (ASM), which thereby generates copious amounts of ceramide to enable enhanced membrane repair. This provides time to complete the process of IEC extrusion. In parallel, we also show that caspase-7 and ASM cleavage are required to clear Chromobacterium violaceum and Listeria monocytogenes after perforin-pore-mediated attack by natural killer cells or cytotoxic T lymphocytes, which normally causes apoptosis in infected hepatocytes. Therefore, caspase-7 is not a conventional executioner but instead is a death facilitator that delays pore-driven lysis so that more-specialized processes, such as extrusion or apoptosis, can be completed before cell death. Cells must put their affairs in order before they die.
Assuntos
Caspase 7 , Perforina , Proteínas de Ligação a Fosfato , Proteínas Citotóxicas Formadoras de Poros , Esfingomielina Fosfodiesterase , Animais , Apoptose , Caspase 7/metabolismo , Chromobacterium/imunologia , Células Epiteliais/citologia , Intestinos/citologia , Células Matadoras Naturais/imunologia , Listeria monocytogenes/imunologia , Camundongos , Organoides , Perforina/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Linfócitos T Citotóxicos/imunologiaRESUMO
Many viruses have evolved structured RNA elements that can influence transcript abundance and translational efficiency, and help evade host immune factors by hijacking cellular machinery during replication. Here, we evaluated the functional impact of sub-genomic flaviviral RNAs (sfRNAs) known to stall exoribonuclease activity, by incorporating these elements into recombinant adeno-associated viral (AAV) genome cassettes. Specifically, sfRNAs from Dengue, Zika, Japanese Encephalitis, Yellow Fever, Murray Valley Encephalitis, and West Nile viruses increased transcript stability and transgene expression compared to a conventional woodchuck hepatitis virus element (WPRE). Further dissection of engineered transcripts revealed that sfRNA elements (i) require incorporation in cis within the 3' untranslated region (UTR) of AAV genomes, (ii) require minimal dumbbell structures to exert the observed effects, and (iii) can stabilize AAV transcripts independent of 5'-3' exoribonuclease 1 (XRN1)-mediated decay. Additionally, preliminary in vivo assessment of AAV vectors bearing sfRNA elements in mice achieved increased transcript abundance and expression in cardiac tissue. Leveraging the functional versatility of engineered viral RNA elements may help improve the potency of AAV vector-based gene therapies. IMPORTANCE: Viral RNA elements can hijack host cell machinery to control stability of transcripts and consequently, infection. Studies that help better understand such viral elements can provide insights into antiviral strategies and also potentially leverage these features for therapeutic applications. In this study, by incorporating structured flaviviral RNA elements into recombinant adeno-associated viral (AAV) vector genomes, we show improved AAV transcript stability and transgene expression can be achieved, with implications for gene transfer.
RESUMO
Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown. Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments revealed that ZCCHC14 binds a small stem-loop in the HAV 5' untranslated RNA possessing a Smaug recognition-like pentaloop to which it recruits TENT4. TENT4 polymerases lengthen and stabilize the 3' poly(A) tails of some cellular and viral mRNAs, but the chemical inhibition of TENT4A/B with the dihydroquinolizinone RG7834 had no impact on the length of the HAV 3' poly(A) tail, stability of HAV RNA, or cap-independent translation of the viral genome. By contrast, RG7834 inhibited the incorporation of 5-ethynyl uridine into nascent HAV RNA, indicating that TENT4A/B function in viral RNA synthesis. Consistent with potent in vitro antiviral activity against HAV (IC50 6.11 nM), orally administered RG7834 completely blocked HAV infection in Ifnar1-/- mice, and sharply reduced serum alanine aminotransferase activities, hepatocyte apoptosis, and intrahepatic inflammatory cell infiltrates in mice with acute hepatitis A. These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.
Assuntos
Proteínas Cromossômicas não Histona , DNA Polimerase Dirigida por DNA , Vírus da Hepatite A , Hepatite A , Proteínas Intrinsicamente Desordenadas , RNA Nucleotidiltransferases , RNA Viral , Replicação Viral , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Proteínas Cromossômicas não Histona/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Hepatite A/tratamento farmacológico , Hepatite A/metabolismo , Hepatite A/virologia , Vírus da Hepatite A/efeitos dos fármacos , Vírus da Hepatite A/genética , Vírus da Hepatite A/fisiologia , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Camundongos , Camundongos Mutantes , RNA Nucleotidiltransferases/metabolismo , RNA Viral/biossíntese , RNA Viral/genética , Receptor de Interferon alfa e beta/genética , Replicação Viral/efeitos dos fármacosRESUMO
Recent discovery of the ribosomal protein (RP) RPL11 interacting with and inhibiting the E3 ubiquitin ligase function of MDM2 established the RP-MDM2-p53 signaling pathway, which is linked to biological events, including ribosomal biogenesis, nutrient availability, and metabolic homeostasis. Mutations in RPs lead to a diverse array of phenotypes known as ribosomopathies in which the role of p53 is implicated. Here, we generated conditional RPL11-deletion mice to investigate in vivo effects of impaired RP expression and its functional connection with p53. While deletion of one Rpl11 allele in germ cells results in embryonic lethality, deletion of one Rpl11 allele in adult mice does not affect viability but leads to acute anemia. Mechanistically, we found RPL11 haploinsufficiency activates p53 in hematopoietic tissues and impedes erythroid precursor differentiation, resulting in insufficient red blood cell development. We demonstrated that reducing p53 dosage by deleting one p53 allele rescues RPL11 haploinsufficiency-induced inhibition of erythropoietic precursor differentiation and restores normal red blood cell levels in mice. Furthermore, blocking the RP-MDM2-p53 pathway by introducing an RP-binding mutation in MDM2 prevents RPL11 haploinsufficiency-caused p53 activation and rescues the anemia in mice. Together, these findings demonstrate that the RP-MDM2-p53 pathway is a critical checkpoint for RP homeostasis and that p53-dependent cell cycle arrest of erythroid precursors is the molecular basis for the anemia phenotype commonly associated with RP deficiency.
Assuntos
Anemia , Proteína Supressora de Tumor p53 , Animais , Camundongos , Anemia/genética , Haploinsuficiência , Mutação , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
IMPORTANCE: Human cytomegalovirus (HCMV) requires inactivation of AKT to efficiently replicate, yet how AKT is shut off during HCMV infection has remained unclear. We show that UL38, an HCMV protein that activates mTORC1, is necessary and sufficient to destabilize insulin receptor substrate 1 (IRS1), a model insulin receptor substrate (IRS) protein. Degradation of IRS proteins in settings of excessive mTORC1 activity is an important mechanism for insulin resistance. When IRS proteins are destabilized, PI3K cannot be recruited to growth factor receptor complexes, and hence, AKT membrane recruitment, a rate limiting step in its activation, fails to occur. Despite its penchant for remodeling host cell signaling pathways, our results reveal that HCMV relies upon a cell-intrinsic negative regulatory feedback loop to inactivate AKT. Given that pharmacological inhibition of PI3K/AKT potently induces HCMV reactivation from latency, our findings also imply that the expression of UL38 activity must be tightly regulated within latently infected cells to avoid spontaneous reactivation.
Assuntos
Citomegalovirus , Proteínas Substratos do Receptor de Insulina , Proteínas Proto-Oncogênicas c-akt , Humanos , Citomegalovirus/fisiologia , Proteínas Substratos do Receptor de Insulina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Estabilidade Proteica , Proteólise , Resistência à Insulina , Retroalimentação Fisiológica , Ativação Viral , Latência ViralRESUMO
We report the synthesis of 2,6-disubstituted pyrazines as potent cell active CSNK2A inhibitors. 4'-Carboxyphenyl was found to be the optimal 2-pyrazine substituent for CSNK2A activity, with little tolerance for additional modification. At the 6-position, modifications of the 6-isopropylaminoindazole substituent were explored to improve selectivity over PIM3 while maintaining potent CSNK2A inhibition. The 6-isopropoxyindole analogue 6c was identified as a nanomolar CSNK2A inhibitor with 30-fold selectivity over PIM3 in cells. Replacement of the 6-isopropoxyindole by isosteric ortho-methoxy anilines, such as 7c, generated analogues with selectivity for CSNK2A over PIM3 and improved the kinome-wide selectivity. The optimized 2,6-disubstituted pyrazines showed inhibition of viral replication consistent with their CSNK2A activity.
Assuntos
Benzoatos , Pirazinas , Relação Estrutura-Atividade , Pirazinas/farmacologia , Antivirais/farmacologiaRESUMO
Host kinases play essential roles in the host cell cycle, innate immune signaling, the stress response to viral infection, and inflammation. Previous work has demonstrated that coronaviruses specifically target kinase cascades to subvert host cell responses to infection and rely upon host kinase activity to phosphorylate viral proteins to enhance replication. Given the number of kinase inhibitors that are already FDA approved to treat cancers, fibrosis, and other human disease, they represent an attractive class of compounds to repurpose for host-targeted therapies against emerging coronavirus infections. To further understand the host kinome response to betacoronavirus infection, we employed multiplex inhibitory bead mass spectrometry (MIB-MS) following MERS-CoV and SARS-CoV-2 infection of human lung epithelial cell lines. Our MIB-MS analyses revealed activation of mTOR and MAPK signaling following MERS-CoV and SARS-CoV-2 infection, respectively. SARS-CoV-2 host kinome responses were further characterized using paired phosphoproteomics, which identified activation of MAPK, PI3K, and mTOR signaling. Through chemogenomic screening, we found that clinically relevant PI3K/mTOR inhibitors were able to inhibit coronavirus replication at nanomolar concentrations similar to direct-acting antivirals. This study lays the groundwork for identifying broad-acting, host-targeted therapies to reduce betacoronavirus replication that can be rapidly repurposed during future outbreaks and epidemics. The proteomics, phosphoproteomics, and MIB-MS datasets generated in this study are available in the Proteomics Identification Database (PRIDE) repository under project identifiers PXD040897 and PXD040901.
Assuntos
COVID-19 , Hepatite C Crônica , Coronavírus da Síndrome Respiratória do Oriente Médio , Humanos , Antivirais/farmacologia , Inibidores de MTOR , Fosfatidilinositol 3-Quinases , SARS-CoV-2 , Replicação Viral , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Serina-Treonina Quinases TORRESUMO
Human progenitor cells (HPCs) support human cytomegalovirus (HCMV) latency, and their differentiation along the myeloid lineage triggers cellular cues that drive reactivation. A key step during HCMV reactivation in latently infected HPCs is reexpression of viral major immediate early (MIE) genes. We recently determined that the major immediate early promoter (MIEP), which is primarily responsible for MIE gene expression during lytic replication, remains silent during reactivation. Instead, alternative promoters in the MIE locus are induced by reactivation stimuli. Here, we find that forkhead family (FOXO) transcription factors are critical for activation of alternative MIE promoters during HCMV reactivation, as mutating FOXO binding sites in alternative MIE promoters decreased HCMV IE gene expression upon reactivation and significantly decreased the production of infectious virus from latently infected primary CD34+ HPCs. These findings establish a mechanistic link by which infected cells sense environmental cues to regulate latency and reactivation, and emphasize the role of contextual activation of alternative MIE promoters as the primary drivers of reactivation.
Assuntos
Citomegalovirus , Fatores de Transcrição Forkhead/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Virais/metabolismo , Citomegalovirus/genética , Citomegalovirus/metabolismo , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/virologia , Genes Precoces/genética , Células HeLa , Humanos , Latência ViralRESUMO
Probabilistic bet hedging, a strategy to maximize fitness in unpredictable environments by matching phenotypic variability to environmental variability, is theorized to account for the evolution of various fate-specification decisions, including viral latency. However, the molecular mechanisms underlying bet hedging remain unclear. Here, we report that large variability in protein abundance within individual herpesvirus virion particles enables probabilistic bet hedging between viral replication and latency. Superresolution imaging of individual virions of the human herpesvirus cytomegalovirus (CMV) showed that virion-to-virion levels of pp71 tegument protein-the major viral transactivator protein-exhibit extreme variability. This super-Poissonian tegument variability promoted alternate replicative strategies: high virion pp71 levels enhance viral replicative fitness but, strikingly, impede silencing, whereas low virion pp71 levels reduce fitness but promote silencing. Overall, the results indicate that stochastic tegument packaging provides a mechanism enabling probabilistic bet hedging between viral replication and latency.
Assuntos
Citomegalovirus/genética , Citomegalovirus/fisiologia , Proteínas Virais/metabolismo , Latência Viral/genética , Latência Viral/fisiologia , Evolução Biológica , Infecções por Citomegalovirus , Regulação Viral da Expressão Gênica , Humanos , Monócitos , Vírion/metabolismo , Replicação ViralRESUMO
Nuclear factor erythroid 2-related factor 2 (NFE2L2, also known as NRF2) is a transcription factor and master regulator of cellular antioxidant response. Aberrantly high NRF2-dependent transcription is recurrent in human cancer, but conversely NRF2 activity diminishes with age and in neurodegenerative and metabolic disorders. Although NRF2-activating drugs are clinically beneficial, NRF2 inhibitors do not yet exist. Here, we describe use of a gain-of-function genetic screen of the kinome to identify new druggable regulators of NRF2 signaling. We found that the under-studied protein kinase brain-specific kinase 2 (BRSK2) and the related BRSK1 kinases suppress NRF2-dependent transcription and NRF2 protein levels in an activity-dependent manner. Integrated phosphoproteomics and RNAseq studies revealed that BRSK2 drives 5'-AMP-activated protein kinase α2 (AMPK) signaling and suppresses the mTOR pathway. As a result, BRSK2 kinase activation suppresses ribosome-RNA complexes, global protein synthesis and NRF2 protein levels. Collectively, our data illuminate the BRSK2 and BRSK1 kinases, in part by functionally connecting them to NRF2 signaling and mTOR. This signaling axis might prove useful for therapeutically targeting NRF2 in human disease.This article has an associated First Person interview with the first author of the paper.
Assuntos
Fator 2 Relacionado a NF-E2 , Receptor EphA5 , Proteínas Quinases Ativadas por AMP/metabolismo , Mutação com Ganho de Função , Humanos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genéticaRESUMO
Reactivation from latency requires reinitiation of viral gene expression and culminates in the production of infectious progeny. The major immediate early promoter (MIEP) of human cytomegalovirus (HCMV) drives the expression of crucial lytic cycle transactivators but is silenced during latency in hematopoietic progenitor cells (HPCs). Because the MIEP has poor activity in HPCs, it is unclear how viral transactivators are expressed during reactivation. It has been presumed that viral gene expression is reinitiated via de-repression of the MIEP. We demonstrate that immediate early transcripts arising from reactivation originate predominantly from alternative promoters within the canonical major immediate early locus. Disruption of these intronic promoters results in striking defects in re-expression of viral genes and viral genome replication in the THP-1 latency model. Furthermore, we show that these promoters are necessary for efficient reactivation in primary CD34+ HPCs. Our findings shift the paradigm for HCMV reactivation by demonstrating that promoter switching governs reactivation from viral latency in a context-specific manner.
Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Regiões Promotoras Genéticas , Ativação Viral , Latência Viral , Células Cultivadas , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Íntrons , Transativadores/genética , Transativadores/metabolismo , Replicação ViralRESUMO
Recent Zika virus (ZIKV) outbreaks and unexpected clinical manifestations of ZIKV infection have prompted an increase in ZIKV-related research. Here, we identify two strain-specific determinants of ZIKV virulence in mice. We found that strain H/PF/2013 caused 100% lethality in Ifnar1-/- mice, whereas PRVABC59 caused no lethality; both strains caused 100% lethality in Ifnar1-/-Ifngr1-/- double-knockout (DKO) mice. Deep sequencing revealed a high-frequency variant in PRVABC59 not present in H/PF/2013: a G-to-T change at nucleotide 1965 producing a Val-to-Leu substitution at position 330 of the viral envelope (E) protein. We show that the V330 variant is lethal on both virus strain backgrounds, whereas the L330 variant is attenuating only on the PRVABC59 background. These results identify a balanced polymorphism in the E protein that is sufficient to attenuate the PRVABC59 strain but not H/PF/2013. The consensus sequences of H/PF/2013 and PRVABC59 differ by 3 amino acids, but these were not responsible for the difference in virulence between the two strains. H/PF/2013 and PRVABC59 differ by an additional 31 noncoding or silent nucleotide changes. We made a panel of chimeric viruses with identical amino acid sequences but nucleotide sequences derived from H/PF/2013 or PRVABC59. We found that 6 nucleotide differences in the 3' quarter of the H/PF/2013 genome were sufficient to confer virulence in Ifnar1-/- mice. Altogether, our work identifies a large and previously unreported difference in virulence between two commonly used ZIKV strains, in two widely used mouse models of ZIKV pathogenesis (Ifnar1-/- and Ifnar1-/- Ifngr1-/- DKO mice).IMPORTANCE Contemporary ZIKV strains are closely related and often used interchangeably in laboratory research. Here, we identify two strain-specific determinants of ZIKV virulence that are evident in only Ifnar1-/- mice but not Ifnar1-/-Ifngr1-/- DKO mice. These results identify a balanced polymorphism in the E protein that is sufficient to attenuate the PRVABC59 strain but not H/PF/2013. We further identify a second virulence determinant in the H/PF/2013 strain, which is driven by the viral nucleotide sequence but not the amino acid sequence. Altogether, our work identifies a large and previously unreported difference in virulence between two commonly used ZIKV strains, in two widely used mouse models of ZIKV pathogenesis. Our results highlight that even very closely related virus strains can produce significantly different pathogenic phenotypes in common laboratory models.
Assuntos
Variação Genética , Proteínas Virais , Infecção por Zika virus , Zika virus , Células A549 , Animais , Chlorocebus aethiops , Feminino , Humanos , Camundongos , Camundongos Knockout , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/imunologia , Receptores de Interferon/genética , Receptores de Interferon/imunologia , Especificidade da Espécie , Células Vero , Proteínas Virais/genética , Proteínas Virais/imunologia , Zika virus/genética , Zika virus/imunologia , Infecção por Zika virus/genética , Infecção por Zika virus/imunologia , Receptor de Interferon gamaRESUMO
Lysosomes maintain immune homeostasis through the degradation of phagocytosed apoptotic debris; however, the signaling events regulating lysosomal maturation remain undefined. In this study, we show that lysosome acidification, key to the maturation process, relies on mTOR complex 2 (mTORC2), activation of caspase-1, and cleavage of Rab39a. Mechanistically, the localization of cofilin to the phagosome recruits caspase-11, which results in the localized activation of caspase-1. Caspase-1 subsequently cleaves Rab39a on the phagosomal membrane, promoting lysosome acidification. Although caspase-1 is critical for lysosome acidification, its activation is independent of inflammasomes and cell death mediated by apoptosis-associated speck-like protein containing a caspase recruitment domain, revealing a role beyond pyroptosis. In lupus-prone murine macrophages, chronic mTORC2 activity decouples the signaling pathway, leaving Rab39a intact. As a result, the lysosome does not acidify, and degradation is impaired, thereby heightening the burden of immune complexes that activate FcγRI and sustain mTORC2 activity. This feedforward loop promotes chronic immune activation, leading to multiple lupus-associated pathologies. In summary, these findings identify the key molecules in a previously unappreciated signaling pathway that promote lysosome acidification. It also shows that this pathway is disrupted in systemic lupus erythematosus.
Assuntos
Caspase 1/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Complexo Antígeno-Anticorpo/metabolismo , Apoptose/fisiologia , Homeostase/fisiologia , Inflamassomos/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose/fisiologia , Fagossomos/metabolismo , Piroptose/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Alphaviruses are mosquito-borne pathogens that cause human diseases ranging from debilitating arthritis to lethal encephalitis. Studies with Sindbis virus (SINV), which causes fever, rash, and arthralgia in humans, and Venezuelan equine encephalitis virus (VEEV), which causes encephalitis, have identified RNA structural elements that play key roles in replication and pathogenesis. However, a complete genomic structural profile has not been established for these viruses. We used the structural probing technique SHAPE-MaP to identify structured elements within the SINV and VEEV genomes. Our SHAPE-directed structural models recapitulate known RNA structures, while also identifying novel structural elements, including a new functional element in the nsP1 region of SINV whose disruption causes a defect in infectivity. Although RNA structural elements are important for multiple aspects of alphavirus biology, we found the majority of RNA structures were not conserved between SINV and VEEV. Our data suggest that alphavirus RNA genomes are highly divergent structurally despite similar genomic architecture and sequence conservation; still, RNA structural elements are critical to the viral life cycle. These findings reframe traditional assumptions about RNA structure and evolution: rather than structures being conserved, alphaviruses frequently evolve new structures that may shape interactions with host immune systems or co-evolve with viral proteins.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , RNA/genética , Sindbis virus/genética , Replicação Viral/genética , Alphavirus/química , Alphavirus/genética , Alphavirus/patogenicidade , Animais , Encefalite/genética , Encefalite/virologia , Vírus da Encefalite Equina Venezuelana/química , Vírus da Encefalite Equina Venezuelana/patogenicidade , Genoma Viral/genética , Cavalos/virologia , Humanos , Conformação de Ácido Nucleico , RNA/química , Sindbis virus/química , Sindbis virus/patogenicidadeRESUMO
Chronic obstructive pulmonary disease (COPD) affects over 65 million individuals worldwide, where α-1-antitrypsin deficiency is a major genetic cause of the disease. The α-1-antitrypsin gene, SERPINA1, expresses an exceptional number of mRNA isoforms generated entirely by alternative splicing in the 5'-untranslated region (5'-UTR). Although all SERPINA1 mRNAs encode exactly the same protein, expression levels of the individual mRNAs vary substantially in different human tissues. We hypothesize that these transcripts behave unequally due to a posttranscriptional regulatory program governed by their distinct 5'-UTRs and that this regulation ultimately determines α-1-antitrypsin expression. Using whole-transcript selective 2'-hydroxyl acylation by primer extension (SHAPE) chemical probing, we show that splicing yields distinct local 5'-UTR secondary structures in SERPINA1 transcripts. Splicing in the 5'-UTR also changes the inclusion of long upstream ORFs (uORFs). We demonstrate that disrupting the uORFs results in markedly increased translation efficiencies in luciferase reporter assays. These uORF-dependent changes suggest that α-1-antitrypsin protein expression levels are controlled at the posttranscriptional level. A leaky-scanning model of translation based on Kozak translation initiation sequences alone does not adequately explain our quantitative expression data. However, when we incorporate the experimentally derived RNA structure data, the model accurately predicts translation efficiencies in reporter assays and improves α-1-antitrypsin expression prediction in primary human tissues. Our results reveal that RNA structure governs a complex posttranscriptional regulatory program of α-1-antitrypsin expression. Crucially, these findings describe a mechanism by which genetic alterations in noncoding gene regions may result in α-1-antitrypsin deficiency.
Assuntos
Processamento Alternativo/genética , Modelos Biológicos , Biossíntese de Proteínas/genética , RNA Mensageiro/química , alfa 1-Antitripsina/genética , Regiões 5' não Traduzidas/genética , Células A549 , Sequência de Bases , Células Hep G2 , Humanos , Mutagênese , Fases de Leitura Aberta/genética , Doença Pulmonar Obstrutiva Crônica/genética , Relação Quantitativa Estrutura-Atividade , Isoformas de RNA/química , Isoformas de RNA/genética , RNA Mensageiro/genética , Deficiência de alfa 1-Antitripsina/genéticaRESUMO
Despite recent advances in therapy, allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative option for a range of high-risk hematologic malignancies. However, acute graft-versus-host disease (aGVHD) continues to limit the long-term success of HSCT, and new therapies are still needed. We previously demonstrated that aGVHD depends on the ability of donor conventional T cells (Tcons) to express the lymph node trafficking receptor, CC-Chemokine Receptor 7 (CCR7). Consequently, we examined the ability of cosalane, a recently identified CCR7 small-molecule antagonist, to attenuate aGVHD in mouse HSCT model systems. Here we show that the systemic administration of cosalane to transplant recipients after allogeneic HSCT did not prevent aGVHD. However, we were able to significantly reduce aGVHD by briefly incubating donor Tcons with cosalane ex vivo before transplantation. Cosalane did not result in Tcon toxicity and did not affect their activation or expansion. Instead, cosalane prevented donor Tcon trafficking into host secondary lymphoid tissues very early after transplantation and limited their subsequent accumulation within the liver and colon. Cosalane did not appear to impair the intrinsic ability of donor Tcons to produce inflammatory cytokines. Furthermore, cosalane-treated Tcons retained their graft-versus-leukemia (GVL) potential and rejected a murine P815 inoculum after transplantation. Collectively, our data indicate that a brief application of cosalane to donor Tcons before HSCT significantly reduces aGVHD in relevant preclinical models while generally sparing beneficial GVL effects, and that cosalane might represent a viable new approach for aGVHD prophylaxis.
Assuntos
Ácido Aurintricarboxílico/análogos & derivados , Doença Enxerto-Hospedeiro/genética , Efeito Enxerto vs Leucemia/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Receptores CCR7/metabolismo , Linfócitos T/metabolismo , Condicionamento Pré-Transplante/métodos , Doença Aguda , Animais , Ácido Aurintricarboxílico/farmacologia , Ácido Aurintricarboxílico/uso terapêutico , Humanos , Camundongos , Doadores de TecidosRESUMO
Circular RNAs (circRNAs) are highly stable, covalently closed RNAs that are regulated in a spatiotemporal manner and whose functions are largely unknown. These molecules have the potential to be incorporated into engineered systems with broad technological implications. Here we describe a switch for inducing back-splicing of an engineered circRNA that relies on the CRISPR endoribonuclease, Csy4, as an activator of circularization. The endoribonuclease activity and 3' end-stabilizing properties of Csy4 are particularly suited for this task. Coexpression of Csy4 and the circRNA switch allows for the removal of downstream competitive splice sites and stabilization of the 5' cleavage product. This subsequently results in back-splicing of the 5' cleavage product into a circRNA that can translate a reporter protein from an internal ribosomal entry site (IRES). Our platform outlines a straightforward approach toward regulating splicing and could find potential applications in synthetic biology as well as in studying the properties of different circRNAs.