RESUMEN
Orf virus (ORFV), a Parapoxvirus in Poxviridae, infects sheep and goats resulting in contagious pustular dermatitis. ORFV is regarded as a promising viral vector candidate for vaccine development and oncolytic virotherapy. Owing to their potential clinical application, safety concerns have become increasingly important. Deletion of either the OV132 (encoding vascular endothelial growth factor, VEGF) or OV112 (encoding the chemokine binding protein, CBP) genes reduced ORFV infectivity, which has been independently demonstrated in the NZ2 and NZ7 strains, respectively. This study revealed that the VEGF and CBP gene sequences of the local strain (TW/Hoping) shared a similarity of 47.01% with NZ2 and 90.56% with NZ7. Due to the high sequence divergence of these two immunoregulatory genes among orf viral strains, their contribution to the pathogenicity of Taiwanese ORFV isolates was comparatively characterized. Initially, two ORFV recombinants were generated, in which either the VEGF or CBP gene was deleted and replaced with the reporter gene EGFP. In vitro assays indicated that both the VEGF-deletion mutant ORFV-VEGFΔ-EGFP and the CBP deletion mutant ORFV-CBPΔ-EGFP were attenuated in cells. In particular, ORFV-VEGFΔ-EGFP significantly reduced plaque size and virus yield compared to ORFV-CBPΔ-EGFP and the wild-type control. Similarly, in vivo analysis revealed no virus yield in the goat skin biopsy infected by ORFV-VEGFΔ-EGFP, and significantly reduced the virus yield of ORFV-CBPΔ-EGFP relative to the wild-type control. These results confirmed the loss of virulence of both deletion mutants in the Hoping strain, whereas the VEGF-deletion mutant was more attenuated than the CBP deletion strain in both cell and goat models. KEY POINTS: ⢠VEGF and CBP genes are crucial in ORFV pathogenesis in the TW/Hoping strain ⢠The VEGF-deletion mutant virus was severely attenuated in both cell culture and animal models ⢠Deletion mutant viruses are advantageous vectors for the development of vaccines and therapeutic regimens.
Asunto(s)
Ectima Contagioso , Virus del Orf , Animales , Ectima Contagioso/patología , Cabras , Virus del Orf/genética , Ovinos , Piel , Factor A de Crecimiento Endotelial Vascular/genética , Genes ViralesRESUMEN
Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine neurodegenerative disease resulting from the misfolding and accumulation of a pathogenic protein, causing cerebellar dysfunction, and this disease currently has no effective treatments. Far-infrared radiation (FIR) has been found to protect the viability of SCA3 cells by preventing mutant ataxin-3 protein aggregation and promoting autophagy. However, this possible treatment still lacks in vivo evidence. This study assessed the effect of FIR therapy on SCA3 in vivo by using a mouse model over 28 weeks. Control mice carried a healthy wild-type ATXN3 allele that had a polyglutamine tract with 15 CAG repeats (15Q), whereas SCA3 transgenic mice possessed an allele with a pathological polyglutamine tract with expanded 84 CAG (84Q) repeats. The results showed that the 84Q SCA3 mice displayed impaired motor coordination, balance abilities, and gait performance, along with the associated loss of Purkinje cells in the cerebellum, compared with the normal 15Q controls; nevertheless, FIR treatment was sufficient to prevent those defects. FIR significantly improved performance in terms of maximal contact area, stride length, and base support in the forepaws, hindpaws, or both. Moreover, FIR treatment supported the survival of Purkinje cells in the cerebellum and promoted the autophagy, as reflected by the induction of autophagic markers, LC3II and Beclin-1, concomitant with the reduction of p62 and ataxin-3 accumulation in cerebellar Purkinje cells, which might partially contribute to the rescue mechanism. In summary, our results reveal that FIR confers therapeutic effects in an SCA3 transgenic animal model and therefore has considerable potential for future clinical use.
Asunto(s)
Cerebelo/patología , Rayos Infrarrojos/uso terapéutico , Enfermedad de Machado-Joseph/patología , Enfermedad de Machado-Joseph/radioterapia , Actividad Motora , Animales , Ataxina-3/genética , Ataxina-3/metabolismo , Autofagia/efectos de la radiación , Cerebelo/metabolismo , Cerebelo/efectos de la radiación , Modelos Animales de Enfermedad , Marcha/efectos de la radiación , Enfermedad de Machado-Joseph/fisiopatología , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/efectos de la radiación , Equilibrio Postural/efectos de la radiación , Distribución AleatoriaRESUMEN
Posttranscriptional mechanisms are important for regulation of cellular and viral gene expression. The presence of the 5' cap structure m(7)G(5')ppp(5')Nm is a general feature of mRNAs that provides protection from exoribonuclease digestion and enhances translation. Vaccinia virus and other poxviruses encode enzymes for both cap synthesis and decapping. Decapping is mediated by two related enzymes, D9 and D10, which are synthesized before and after viral DNA replication, respectively. The timing of D10 synthesis correlates better with the shutdown of host gene expression, and deletion of this gene has been shown to cause persistence of host and viral mRNAs in infected cells. Here, we constructed specific mutant viruses in which translation of D10 was prevented by stop codons or activity of D10 was abrogated by catalytic site mutations, without other genomic alterations. Both mutants formed plaques of normal size and replicated to similar extents as the parental virus in monkey epithelial cells and mouse embryonic fibroblasts. The synthesis of viral proteins was slightly delayed, and cellular and viral mRNAs persisted longer in cells infected with the mutants compared to either the parental virus or clonal revertant. Despite the mild effects in vitro, both mutants were more attenuated than the revertants in intranasal and intraperitoneal mouse models, and less infectious virus was recovered from organs. In addition, there was less lung histopathology following intranasal infection with mutant viruses. These data suggest that the D10 decapping enzyme may help restrict antiviral responses by accelerating host mRNA degradation during poxvirus infection.
Asunto(s)
Nucleotidasas/metabolismo , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Virus Vaccinia/enzimología , Proteínas Virales/metabolismo , Animales , Línea Celular , Cricetinae , Ratones , Ratones Endogámicos C57BL , Virus Vaccinia/genética , Virus Vaccinia/patogenicidad , Virulencia , Replicación ViralRESUMEN
The human scavenger decapping enzyme, DcpS, functions to hydrolyze the resulting cap structure following cytoplasmic mRNA decay yet is, surprisingly, a nuclear protein by immunofluorescence. Here, we show that DcpS is a nucleocytoplasmic shuttling protein that contains separable nuclear import and Crm-1-dependent export signals. We postulated that the presence of DcpS in both cellular compartments and its ability to hydrolyze cap structure may impact other cellular events dependent on cap-binding proteins. An shRNA-engineered cell line with markedly diminished DcpS levels led to a corresponding reduction in cap-proximal intron splicing of a reporter minigene and endogenous genes. The impaired cap catabolism and resultant imbalanced cap concentrations were postulated to sequester the cap-binding complex (CBC) from its normal splicing function. In support of this explanation, DcpS efficiently displaced the nuclear cap-binding protein Cbp20 from cap structure, and complementation with Cbp20 reversed the reduced splicing, indicating that modulation of splicing by DcpS is mediated through Cbp20. Our studies demonstrate that the significance of DcpS extends beyond its well-characterized role in mRNA decay and involves a broader range of functions in RNA processing including nuclear pre-mRNA splicing.
Asunto(s)
Endorribonucleasas/metabolismo , Precursores del ARN/metabolismo , Empalme del ARN , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Línea Celular , Núcleo Celular/química , Núcleo Celular/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Endorribonucleasas/análisis , Endorribonucleasas/genética , Células HeLa , Humanos , Intrones , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Señales de Exportación Nuclear/genética , Señales de Localización Nuclear/análisis , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Empalme del ARN/genética , RatasRESUMEN
RBM4 participates in cell differentiation by regulating tissue-specific alternative pre-mRNA splicing. RBM4 also has been implicated in neurogenesis in the mouse embryonic brain. Using mouse embryonal carcinoma P19 cells as a neural differentiation model, we observed a temporal correlation between RBM4 expression and a change in splicing isoforms of Numb, a cell-fate determination gene. Knockdown of RBM4 affected the inclusion/exclusion of exons 3 and 9 of Numb in P19 cells. RBM4-deficient embryonic mouse brain also exhibited aberrant splicing of Numb pre-mRNA. Using a splicing reporter minigene assay, we demonstrated that RBM4 promoted exon 3 inclusion and exon 9 exclusion. Moreover, we found that RBM4 depletion reduced the expression of the proneural gene Mash1, and such reduction was reversed by an RBM4-induced Numb isoform containing exon 3 but lacking exon 9. Accordingly, induction of ectopic RBM4 expression in neuronal progenitor cells increased Mash1 expression and promoted cell differentiation. Finally, we found that RBM4 was also essential for neurite outgrowth from cortical neurons in vitro. Neurite outgrowth defects of RBM4-depleted neurons were rescued by RBM4-induced exon 9-lacking Numb isoforms. Therefore our findings indicate that RBM4 modulates exon selection of Numb to generate isoforms that promote neuronal cell differentiation and neurite outgrowth.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo , Animales , Diferenciación Celular/genética , Exones , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Neuritas/metabolismo , Neurogénesis , Proyección Neuronal , Neuronas/metabolismo , Cultivo Primario de Células , Isoformas de Proteínas/metabolismo , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Células Madre/metabolismoRESUMEN
Poxvirus replication involves synthesis of double-stranded RNA (dsRNA), which can trigger antiviral responses by inducing phosphorylation-mediated activation of protein kinase R (PKR) and stimulating 2'5'-oligoadenylate synthetase (OAS). PKR inactivates the translation initiation factor eIF2α via phosphorylation, while OAS induces the endonuclease RNase L to degrade RNA. We show that poxvirus decapping enzymes D9 and D10, which remove caps from mRNAs, inhibit these antiviral responses by preventing dsRNA accumulation. Catalytic site mutations of D9 and D10, but not of either enzyme alone, halt vaccinia virus late protein synthesis and inhibit virus replication. Infection with the D9-D10 mutant was accompanied by massive mRNA reduction, cleavage of ribosomal RNA, and phosphorylation of PKR and eIF2α that correlated with a â¼ 15-fold increase in dsRNA compared to wild-type virus. Additionally, mouse studies show extreme attenuation of the mutant virus. Thus, vaccinia virus decapping, in addition to targeting mRNAs for degradation, prevents dsRNA accumulation and anti-viral responses.
Asunto(s)
Endorribonucleasas/metabolismo , Inmunidad Innata , ARN Bicatenario/metabolismo , Virus Vaccinia/enzimología , Virus Vaccinia/inmunología , Replicación Viral , 2',5'-Oligoadenilato Sintetasa/metabolismo , Animales , Línea Celular , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , Virus Vaccinia/crecimiento & desarrollo , Virus Vaccinia/fisiología , Proteínas Virales/metabolismo , Virulencia , eIF-2 Quinasa/metabolismoRESUMEN
The African Swine Fever Virus (ASFV) encodes a single Nudix enzyme in its genome, termed the g5R protein (g5Rp). Nudix phosphohydrolases cleave a variety of substrates, such as nucleotides and diphosphoinositol polyphosphates. Previously, ASFV g5Rp was shown to hydrolyze diphosphoinositol polyphosphates and GTP, but was unable to cleave methylated mRNA cap analogues. In vaccinia virus (VACV), a distant relative of ASFV, the D9 and D10 Nudix enzymes were shown to cleave the mRNA cap, but only when the cap was attached to an RNA body. Here, we show that recombinant ASFV g5Rp hydrolyzes the mRNA cap when tethered to an RNA moiety, liberating m(7)GDP as a product. Mutations in the Nudix motif abolished mRNA decapping activity, confirming that g5Rp was responsible for cap cleavage. The decapping activity of g5Rp was potently inhibited by excess uncapped RNA but not by methylated cap analogues, suggesting that substrate recognition occurs by RNA binding.
Asunto(s)
Virus de la Fiebre Porcina Africana/fisiología , Endorribonucleasas/metabolismo , Pirofosfatasas/metabolismo , ARN Mensajero/metabolismo , Proteínas Virales/metabolismo , Endorribonucleasas/genética , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Hidrolasas NudixRESUMEN
Decapping is an important process in the control of eukaryotic mRNA degradation. The scavenger decapping enzyme DcpS functions to clear the cell of cap structure following decay of the RNA body by catalyzing the hydrolysis of m(7)GpppN to m(7)Gp and ppN. Structural analysis has revealed that DcpS is a dimeric protein with a domain-swapped amino terminus. The protein dimer contains two cap binding/hydrolysis sites and displays a symmetric structure with both binding sites in the open conformation in the ligand-free state and an asymmetric conformation with one site open and one site closed in the ligand-bound state. The structural data are suggestive of a dynamic decapping mechanism where each monomer could alternate between an open and closed state. Using transient state kinetic studies, we show that both the rate-limiting step and rate of decapping are regulated by cap substrate. A regulatory mechanism is established by the intrinsic domain-swapped structure of the DcpS dimer such that the decapping reaction is very efficient at low cap substrate concentrations yet regulated with excess cap substrate. These data provide biochemical evidence to verify experimentally a dynamic and mutually exclusive cap hydrolysis activity of the two cap binding sites of DcpS and provide key insights into its regulation.
Asunto(s)
Endorribonucleasas/química , Endorribonucleasas/fisiología , Caperuzas de ARN/química , Sitios de Unión , Bioquímica/métodos , Dimerización , Humanos , Hidrólisis , Cinética , Ligandos , Modelos Biológicos , Mutación , Conformación Proteica , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas Recombinantes/químicaRESUMEN
The modulation of mRNA decay is a critical determinant in the regulation of gene expression. mRNAs in eukaryotes are primarily degraded by two major exonucleolytic pathways: the 5' to 3'-and the 3' to 5'-pathways, both of which are initiated by removal of the polyadenylated (poly(A)) tail. Hydrolysis of the 5'-cap structure, termed decapping, is a key step in the demise of mRNA. Two major decapping enzymes with distinct activities and substrate requirements have been identified. Dcp2 hydrolyzes the cap structure on an intact mRNA in the 5' to 3'-decay pathway; Dcp2 scavenges the residual cap oligonucleotide resulting from the 3' to 5'-decay pathway, as well as hydrolyzes the decapping product generated by Dcp2. In this chapter, we describe the methods for monitoring Dcp2 and DcpS decapping activities of bacterially expressed and endogenous human decapping enzymes.
Asunto(s)
Técnicas Genéticas , Caperuzas de ARN/metabolismo , Animales , Tampones (Química) , Endorribonucleasas/metabolismo , Activación Enzimática , Humanos , Caperuzas de ARN/análisis , Caperuzas de ARN/genéticaRESUMEN
Spinal muscular atrophy (SMA) is caused by deletion or mutation of both copies of the SMN1 gene, which produces an essential protein known as SMN. The severity of SMA is modified by variable copy number of a second gene,SMN2, which produces an mRNA that is incorrectly spliced with deletion of the last exon. We described previously the discovery of potent C5-substituted quinazolines that increase SMN2 gene expression by 2-fold. Discovery of potent SMN2 promoter inducers relied on a cellular assay without knowledge of the molecular target. Using protein microarray scanning with a radiolabeled C5-substituted quinazoline probe, we identified the scavenger decapping enzyme, DcpS, as a potential binder. We show that the C5-substituted quinazolines potently inhibit DcpS decapping activity and that the potency of inhibition correlates with potency forSMN2 promoter induction. Binding of C5-substituted quinazolines to DcpS holds the enzyme in an open, catalytically incompetent conformation. DcpS is a nuclear shuttling protein that binds and hydrolyzes the m(7)GpppN mRNA cap structure and a modulator of RNA metabolism. Therefore DcpS represents a novel therapeutic target for modulating gene expression by a small molecule.
Asunto(s)
Endorribonucleasas/antagonistas & inhibidores , Atrofia Muscular Espinal/tratamiento farmacológico , Quinazolinas/farmacología , Sistemas de Liberación de Medicamentos , Humanos , Unión Proteica , Conformación Proteica/efectos de los fármacosRESUMEN
Complete removal of residual N-7 guanine cap from degraded messenger RNA is necessary to prevent accumulation of intermediates that might interfere with RNA processing, export, and translation. The human scavenger decapping enzyme, DcpS, catalyzes residual cap hydrolysis following mRNA degradation, releasing N-7 methyl guanosine monophosphate and 5'-diphosphate terminated cap or mRNA products. DcpS structures bound to m(7)GpppG or m(7)GpppA reveal an asymmetric DcpS dimer that simultaneously creates an open nonproductive DcpS-cap complex and a closed productive DcpS-cap complex that alternate via 30 A domain movements. Structural and biochemical analysis suggests an autoregulatory mechanism whereby premature decapping mRNA is prevented by blocking the conformational changes that are required to form a closed productive active site capable of cap hydrolysis.
Asunto(s)
Endorribonucleasas/química , Endorribonucleasas/metabolismo , Conformación Proteica , Caperuzas de ARN/química , Caperuzas de ARN/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Endorribonucleasas/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , Alineación de SecuenciaRESUMEN
Eukaryotic cells primarily utilize exoribonucleases and decapping enzymes to degrade their mRNA. Two major decapping enzymes have been identified. The hDcp2 protein catalyzes hydrolysis of the 5' cap linked to an RNA moiety, whereas the scavenger decapping enzyme, DcpS, functions on a cap structure lacking the RNA moiety. DcpS is a member of the histidine triad (HIT) family of hydrolases and catalyzes the cleavage of m7GpppN. HIT proteins are homodimeric and contain two conserved 100-amino-acid HIT fold domains with independent active sites that are each sufficient to bind and hydrolyze cognate substrates. We carried out a functional characterization of the DcpS enzyme and demonstrate that unlike previously described HIT proteins, DcpS is a modular protein that requires both the core HIT fold at the carboxyl-terminus and sequences at the amino-terminus of the protein for cap binding and hydrolysis. Interestingly, DcpS can efficiently compete for and hydrolyze the cap structure even in the presence of excess eIF4E, implying that DcpS could function to alleviate the accumulation of complexes between eIF4E and cap structure that would otherwise accumulate following mRNA decay. Using immunofluorescence microscopy, we demonstrate that DcpS is predominantly a nuclear protein, with low levels of detected protein in the cytoplasm. Furthermore, analysis of the endogenous hDcp2 protein reveals that in addition to the cytoplasmic foci, it is also present in the nucleus. These data reveal that both decapping enzymes are contained in the nuclear compartment, indicating that they may fulfill a greater function in the nucleus than previously appreciated.