RESUMO
Pathogen immune responses are profoundly attenuated in fetuses and premature infants, yet the mechanisms underlying this developmental immaturity remain unclear. Here we show transcriptomic, metabolic and polysome profiling and find that monocytes isolated from infants born early in gestation display perturbations in PPAR-γ-regulated metabolic pathways, limited glycolytic capacity and reduced ribosomal activity. These metabolic changes are linked to a lack of translation of most cytokines and of MALT1 signalosome genes essential to respond to the neonatal pathogen Candida. In contrast, they have little impact on house-keeping phagocytosis functions. Transcriptome analyses further indicate a role for mTOR and its putative negative regulator DNA Damage Inducible Transcript 4-Like in regulating these metabolic constraints. Our results provide a molecular basis for the broad susceptibility to multiple pathogens in these infants, and suggest that the fetal immune system is metabolically programmed to avoid energetically costly, dispensable and potentially harmful immune responses during ontogeny.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Imunidade Inata , Monócitos/imunologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/imunologia , PPAR gama/imunologia , Fatores de Transcrição/imunologia , Adulto , Proteína 10 de Linfoma CCL de Células B/deficiência , Proteína 10 de Linfoma CCL de Células B/genética , Proteína 10 de Linfoma CCL de Células B/imunologia , Proteínas Adaptadoras de Sinalização CARD/deficiência , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/imunologia , Candida albicans/imunologia , Candida parapsilosis/imunologia , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Interleucinas/deficiência , Interleucinas/genética , Interleucinas/imunologia , Lectinas Tipo C/deficiência , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Lipopolissacarídeos/farmacologia , Análise em Microsséries , Monócitos/citologia , Monócitos/efeitos dos fármacos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/deficiência , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , PPAR gama/deficiência , PPAR gama/genética , Cultura Primária de Células , Biossíntese de Proteínas/imunologia , Serina-Treonina Quinases TOR/deficiência , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/imunologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Transcriptoma/imunologia , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
All viruses must successfully harness the host translational apparatus and divert it towards viral protein synthesis. Dicistroviruses use an unusual internal ribosome entry site (IRES) mechanism whereby the IRES adopts a three-pseudoknot structure that accesses the ribosome tRNA binding sites to directly recruit the ribosome and initiate translation from a non-AUG start site. A subset of dicistroviruses, including the honey bee Israeli acute paralysis virus (IAPV), encode an extra stem-loop (SLVI) 5' -adjacent to the IGR IRES. Previously, the function of this additional stem-loop is unknown. Here, we provide mechanistic and functional insights into the role of SLVI in IGR IRES translation and in virus infection. Biochemical analyses of a series of mutant IRESs demonstrated that SLVI does not function in ribosome recruitment but is required for proper ribosome positioning on the IRES to direct translation. Using a chimeric infectious clone derived from the related Cricket paralysis virus, we showed that the integrity of SLVI is important for optimal viral translation and viral yield. Based on structural models of ribosome-IGR IRES complexes, the SLVI is predicted to be in the vicinity of the ribosome E site. We propose that SLVI of IAPV IGR IRES functionally mimics interactions of an E-site tRNA with the ribosome to direct positioning of the tRNA-like domain of the IRES in the A site.IMPORTANCEViral internal ribosome entry sites are RNA elements and structures that allow some positive-sense monopartite RNA viruses to hijack the host ribosome to start viral protein synthesis. We demonstrate that a unique stem-loop structure is essential for optimal viral protein synthesis and for virus infection. Biochemical evidence shows that this viral stem-loop RNA structure impacts a fundamental property of the ribosome to start protein synthesis.
Assuntos
Abelhas/virologia , Dicistroviridae/genética , Sítios Internos de Entrada Ribossomal/fisiologia , Biossíntese de Proteínas , Ribossomos/química , Animais , DNA Intergênico/genética , Dicistroviridae/química , Dicistroviridae/metabolismo , Drosophila/genética , Mutação , RNA de Transferência/fisiologia , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Viral genomes are compact and encode a limited number of proteins. Because they do not encode components of the translational machinery, viruses exhibit an absolute dependence on the host ribosome and factors for viral messenger RNA (mRNA) translation. In order to recruit the host ribosome, viruses have evolved unique strategies to either outcompete cellular transcripts that are efficiently translated by the canonical translation pathway or to reroute translation factors and ribosomes to the viral genome. Furthermore, viruses must evade host antiviral responses and escape immune surveillance. This review focuses on some recent major findings that have revealed unconventional strategies that viruses utilize, which include usurping the host translational machinery, modulating canonical translation initiation factors to specifically enhance or repress overall translation for the purpose of viral production, and increasing viral coding capacity. The discovery of these diverse viral strategies has provided insights into additional translational control mechanisms and into the viral host interactions that ensure viral protein synthesis and replication.
Assuntos
Regulação Viral da Expressão Gênica , Biossíntese de Proteínas , Vírus/genética , Interações Hospedeiro-ParasitaRESUMO
The dicistrovirus intergenic internal ribosome entry site (IGR IRES) directly recruits the ribosome and initiates translation using a non-AUG codon. A subset of IGR IRESs initiates translation in either of two overlapping open reading frames (ORFs), resulting in expression of the 0 frame viral structural polyprotein and an overlapping +1 frame ORFx. A U-G base pair adjacent to the anticodon-like pseudoknot of the IRES directs +1 frame translation. Here, we show that the U-G base pair is not absolutely required for +1 frame translation. Extensive mutagenesis demonstrates that 0 and +1 frame translation can be uncoupled. Ribonucleic acid (RNA) structural probing analyses reveal that the mutant IRESs adopt distinct conformations. Toeprinting analysis suggests that the reading frame is selected at a step downstream of ribosome assembly. We propose a model whereby the IRES adopts conformations to occlude the 0 frame aminoacyl-tRNA thereby allowing delivery of the +1 frame aminoacyl-tRNA to the A site to initiate translation of ORFx. This study provides a new paradigm for programmed recoding mechanisms that increase the coding capacity of a viral genome.
Assuntos
Fases de Leitura Aberta , Iniciação Traducional da Cadeia Peptídica , RNA Viral/química , Pareamento de Bases , Dicistroviridae/genética , Células HeLa , Humanos , Mutação , Conformação de Ácido Nucleico , Ribossomos/metabolismoRESUMO
Dyskerin (encoded by the DKC1 locus) is the pseudouridine synthase responsible for the modification of noncoding RNA. Dyskerin is also an obligate member of the telomerase enzyme, and participates in the biogenesis of telomerase. Genetic lesions at the DKC1 locus are associated with X-linked dyskeratosis congenita (X-DC) and the Hoyeraal-Hreidarsson Syndrome (HHS). Both syndromes have been linked to deficient telomere maintenance, but little is known about the RNA modification activities of dyskerin in X-DC and HHS cells. To evaluate whether X-DC-associated dyskerin mutations affect the modification or function of ribosomal RNA, we studied five telomerase-rescued X-DC cells (X-DC(T) ). Our data revealed a small reproducible loss of pseudouridines in mature rRNA in two X-DC variants. However, we found no difference in protein synthesis between telomerized wild-type (WT(T) ) and X-DC(T) cells, with an internal ribosomal entry site translation assay, or by measuring total protein synthesis in live cells. X-DC(T) cells and WT(T) cells also exhibited similar tolerances to ionizing radiation and endoplasmic reticulum stress. Despite the loss in rRNA pseudouridine modification, functional perturbations from these changes are secondary to the telomere maintenance defects of X-DC. Our data show that telomere dysfunction is the primary and unifying etiology of X-DC.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Disceratose Congênita/genética , Disceratose Congênita/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Biossíntese de Proteínas , RNA Ribossômico/genética , Linhagem Celular , Sistema Livre de Células , Fibroblastos/metabolismo , Expressão Gênica , Estudos de Associação Genética , Humanos , Mutação , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Subunidades Ribossômicas/metabolismo , Índice de Gravidade de Doença , Estresse Fisiológico , Telomerase/genética , Telomerase/metabolismo , Telômero/metabolismoRESUMO
Internal ribosome entry sites are RNA elements that mediate translation in a cap-independent manner. A subset of positive strand RNA viruses utilize an IRES mechanism as a viral strategy to ensure efficient viral protein synthesis. IRES elements vary in sequence, structure, and factor requirements between virus families. Here, we describe methods to determine IRES activity and approaches to study the regulation and function of IRES-mediated translation both in vitro and in vivo. Finally, we describe a new IRES-directed reporter system which exploits the 2A 'self-cleavage' or 'stop-go' peptide for optimal detection of IRES activity.
Assuntos
Técnicas Genéticas , Biossíntese de Proteínas/genética , Vírus de RNA/genética , Ribossomos/genética , Ribossomos/virologia , Animais , Sequência de Bases , Humanos , Dados de Sequência MolecularRESUMO
The intergenic region internal ribosome entry site (IGR IRES) of the Dicistroviridae family adopts an overlapping triple pseudoknot structure to directly recruit the 80S ribosome in the absence of initiation factors. The pseudoknot I (PKI) domain of the IRES mimics a tRNA-like codon:anticodon interaction in the ribosomal P site to direct translation initiation from a non-AUG initiation codon in the A site. In this study, we have performed a comprehensive mutational analysis of this region to delineate the molecular parameters that drive IRES translation. We demonstrate that IRES-mediated translation can initiate at an alternate adjacent and overlapping start site, provided that basepairing interactions within PKI remain intact. Consistent with this, IGR IRES translation tolerates increases in the variable loop region that connects the anticodon- and codon-like elements within the PKI domain, as IRES activity remains relatively robust up to a 4-nucleotide insertion in this region. Finally, elements from an authentic tRNA anticodon stem-loop can functionally supplant corresponding regions within PKI. These results verify the importance of the codon:anticodon interaction of the PKI domain and further define the specific elements within the tRNA-like domain that contribute to optimal initiator Met-tRNA(i)-independent IRES translation.
Assuntos
Anticódon/metabolismo , Códon/metabolismo , Dicistroviridae/genética , Iniciação Traducional da Cadeia Peptídica/fisiologia , Ribossomos/metabolismo , Pareamento de Bases , Análise Mutacional de DNA , Primers do DNA/genética , DNA Intergênico/genética , Dicistroviridae/fisiologia , Conformação de Ácido Nucleico , RNA de Transferência/genética , Ribossomos/genéticaRESUMO
The dicistrovirus is a positive-strand single-stranded RNA virus that possesses two internal ribosome entry sites (IRES) that direct translation of distinct open reading frames encoding the viral structural and nonstructural proteins. Through an unusual mechanism, the intergenic region (IGR) IRES responsible for viral structural protein expression mimics a tRNA to directly recruit the ribosome and set the ribosome into translational elongation. In this study, we explored the mechanism of host translational shutoff in Drosophila S2 cells infected by the dicistrovirus, cricket paralysis virus (CrPV). CrPV infection of S2 cells results in host translational shutoff concomitant with an increase in viral protein synthesis. CrPV infection resulted in the dissociation of eukaryotic translation initiation factor 4G (eIF4G) and eIF4E early in infection and the induction of deIF2alpha phosphorylation at 3 h postinfection, which lags after the initial inhibition of host translation. Forced dephosphorylation of deIF2alpha by overexpression of dGADD34, which activates protein phosphatase I, did not prevent translational shutoff nor alter virus production, demonstrating that deIF2alpha phosphorylation is dispensable for host translational shutoff. However, premature induction of deIF2alpha phosphorylation by thapsigargin treatment early in infection reduced viral protein synthesis and replication. Finally, translation mediated by the 5' untranslated region (5'UTR) and the IGR IRES were resistant to impairment of eIF4F or eIF2 in translation extracts. These results support a model by which the alteration of the deIF4F complex contribute to the shutoff of host translation during CrPV infection, thereby promoting viral protein synthesis via the CrPV 5'UTR and IGR IRES.