RESUMEN
Coronavirus replication and transcription are processes mediated by a protein complex, with the RNA-dependent RNA polymerase (RdRp) as a main component. Proteomic analysis of highly purified transmissible gastroenteritis virus showed the RdRp to be a component of the viral particles. This finding was confirmed by Western blotting, immunofluorescence, and immunoelectron microscopy analyses. Interestingly, the replicase nonstructural proteins 2, 3, and 8 colocalized with the RdRp in the viral factories and were also incorporated into the virions.
Asunto(s)
Gastroenteritis Porcina Transmisible/virología , ARN Polimerasa Dependiente del ARN/metabolismo , Virus de la Gastroenteritis Transmisible/fisiología , Proteínas no Estructurales Virales/metabolismo , Virión/fisiología , Ensamble de Virus , Animales , ARN Polimerasa Dependiente del ARN/genética , Porcinos , Virus de la Gastroenteritis Transmisible/enzimología , Virus de la Gastroenteritis Transmisible/genética , Proteínas no Estructurales Virales/genética , Virión/enzimología , Virión/genética , Replicación ViralRESUMEN
Arabidopsis thaliana S-Phase Kinase-Associated Protein 2A (SKP2A) is an F-box protein that regulates the proteolysis of cell cycle transcription factors. The plant hormone auxin regulates multiple aspects of plant growth and development, including cell division. We found that auxin induces the ubiquitin-dependent degradation of SKP2A both in vivo and in vitro, suggesting that this hormone acts as a signal to trigger SKP2A proteolysis. In this article, we show that auxin binds directly and specifically to SKP2A. By TIR1-based superposition and docking analyzes, we identified an auxin binding site in SKP2A. Mutations in this binding site reduce the ability of SKP2A to bind to auxin and generate nondegradable SKP2A forms. In addition, these non-auxin binding proteins are unable to promote E2FC/DPB degradation in vivo or to induce cell division in the root meristem. Auxin binds to TIR1 to promote its interaction with the auxin/indole-3-acetic acid target proteins. Here, we show that auxin also enhanced the interaction between SKP2A and DPB. Finally, a mutation in SKP2A leads to auxin-resistant root growth, an effect that is additive with the tir1-1 phenotype. Thus, our data indicate that SKP2A is an auxin binding protein that connects auxin signaling with cell division.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Proteínas F-Box/metabolismo , Ácidos Indolacéticos/metabolismoRESUMEN
BACKGROUND: The fight against the current coronavirus disease 2019 (COVID-19) pandemic has created a huge demand of biotechnological, pharmaceutical, research and sanitary materials at unprecedented scales. One of the most urgent demands affects the diagnostic tests. The growing need for rapid and accurate laboratory diagnostic tests requires the development of biotechnological processes aimed at producing reagents able to cope with this demand in a scalable, cost-effective manner, with rapid turnaround times. This is particularly applicable to the antigens employed in serological tests. Recombinant protein expression using plants as biofactories is particularly suitable for mass production of protein antigens useful in serological diagnosis, with a neat advantage in economic terms. METHODS: We expressed a large portion of the nucleoprotein (N) derived from SARS-CoV-2 in Nicotiana benthamiana plants. After purification, the recombinant N protein obtained was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to SARS-CoV-2 in human sera. To validate the ELISA, a panel of 416 sera from exposed personnel at essential services in Madrid City Council were tested, and the results compared to those obtained by another ELISA, already validated, used as reference. Furthermore, a subset of samples for which RT-PCR results were available were used to confirm sensitivity and specificity of the test. RESULTS: The performance of the N protein expressed in plants as antigen in serologic test for SARS-CoV-2 antibody detection was shown to be highly satisfactory, with calculated diagnostic sensitivity of 96.41% (95% CI: 93.05-98.44) and diagnostic specificity of 96.37 (95% CI: 93.05-98.44) as compared to the reference ELISA, with a kappa (K) value of 0.928 (95% CI:0.892-0.964). Furthermore, the ELISA developed with plant-derived N antigen detected SARS-CoV-2 antibodies in 84 out of 93 sera from individuals showing RT-PCR positive results (86/93 for the reference ELISA). CONCLUSION: This study demonstrates that the N protein part derived from SARS-CoV-2 expressed in plants performs as a perfectly valid antigen for use in COVID-19 diagnosis. Furthermore, our results support the use of this plant platform for expression of recombinant proteins as reagents for COVID-19 diagnosis. This platform stands out as a convenient and advantageous production system, fit-for-purpose to cope with the current demand of this type of biologicals in a cost-effective manner, making diagnostic kits more affordable.
RESUMEN
Coordination between cell division and cell differentiation is crucial for growth and development of eukaryotic organisms. Progression through the different phases of cell division requires the specific degradation of proteins through the ubiquitin/proteasome 26S (Ub/26S) pathway. In plants, this pathway plays a key role in controlling several developmental processes and responses, including cell proliferation. SKP2A, an F-box protein, regulates the stability of the cell division E2FC-DPB transcription factor. Here, we show that the SKP2A forms a Skp, Cullin containing (SCF) complexin vivo that has E3 ubiquitin ligase activity. Interestingly, SKP2A is degraded through the Ub/26S pathway, and auxin regulates such degradation. SKP2A positively regulates cell division, at least in part by degrading the E2FC/DPB transcription repressor. Plants that overexpress SKP2A increase the number of cells in G2/M, reduce the level of ploidy and develop a higher number of lateral root primordia. Taken together, our results indicate that SKP2A is a positive regulator of cell division, and its stability is controlled by auxin-dependent degradation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas F-Box/metabolismo , Ubiquitina/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , División Celular , Proteínas F-Box/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Ácidos Indolacéticos/metabolismo , Meristema/citología , Meristema/metabolismo , Plantas Modificadas Genéticamente , Proteínas Ligasas SKP Cullina F-box/metabolismoRESUMEN
Zika virus (ZIKV) infection is currently one of the major concerns in human public health due to its association with neurological disorders. Intensive effort has been implemented for the treatment of ZIKV, however there are not currently approved vaccines or antivirals available to combat ZIKV infection. In this sense, the identification of virulence factors associated with changes in ZIKV virulence could help to develop safe and effective countermeasures to treat ZIKV or to prevent future outbreaks. Here, we have compared the virulence of two related ZIKV strains from the recent outbreak in Brazil (2015), Rio Grande do Norte Natal (RGN) and Paraiba. In spite of both viruses being identified in the same period of time and region, significant differences in virulence and replication were observed using a validated mouse model of ZIKV infection. While ZIKV-RGN has a 50% mouse lethal dose (MLD50) of ~105 focus forming units (FFUs), ZIKV-Paraiba infection resulted in 100% of lethality with less than 10 FFUs. Combining deep-sequencing analysis and our previously described infectious ZIKV-RGN cDNA clone, we identified a natural polymorphism in the non-structural protein 2 A (NS2A) that increase the virulence of ZIKV. Moreover, results demonstrate that the single amino acid alanine to valine substitution at position 117 (A117V) in the NS2A was sufficient to convert the attenuated rZIKV-RGN in a virulent Paraiba-like virus (MLD50 < 10 FFU). The mechanism of action was also evaluated and data indicate that substitution A117V in ZIKV NS2A protein reduces host innate immune responses and viral-induced apoptosis in vitro. Therefore, amino acid substitution A117V in ZIKV NS2A could be used as a genetic risk-assessment marker for future ZIKV outbreaks.
Asunto(s)
Polimorfismo Genético , Proteínas no Estructurales Virales/genética , Infección por el Virus Zika/virología , Virus Zika/fisiología , Sustitución de Aminoácidos , Animales , Apoptosis , Línea Celular , Modelos Animales de Enfermedad , Femenino , Genoma Viral , Genómica/métodos , Interacciones Huésped-Patógeno , Inmunidad Innata , Ratones , Virulencia/genética , Replicación Viral , Infección por el Virus Zika/inmunologíaRESUMEN
The recent outbreaks of Zika virus (ZIKV), its association with Guillainâ»Barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat ZIKV disease. In this respect, infectious clones constitute excellent tools to accomplish these goals. However, flavivirus infectious clones are often difficult to work with due to the toxicity of some flavivirus sequences in bacteria. To bypass this problem, several alternative approaches have been applied for the generation of ZIKV clones including, among others, in vitro ligation, insertions of introns and using infectious subgenomic amplicons. Here, we report a simple and novel DNA-launched approach based on the use of a bacterial artificial chromosome (BAC) to generate a cDNA clone of Rio Grande do Norte Natal ZIKV strain. The sequence was identified from the brain tissue of an aborted fetus with microcephaly. The BAC clone was fully stable in bacteria and the infectious virus was efficiently recovered in Vero cells through direct delivery of the cDNA clone. The rescued virus yielded high titers in Vero cells and was pathogenic in a validated mouse model (A129 mice) of ZIKV infection. Furthermore, using this infectious clone we have generated a mutant ZIKV containing a single amino acid substitution (A175V) in the NS2A protein that presented reduced viral RNA synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with ZIKV wild-type. This BAC approach provides a stable and reliable reverse genetic system for ZIKV that will help to identify viral determinants of virulence and facilitate the development of vaccine and therapeutic strategies.
Asunto(s)
Proteínas no Estructurales Virales/genética , Proteínas Virales/genética , Infección por el Virus Zika/patología , Infección por el Virus Zika/virología , Virus Zika/genética , Virus Zika/patogenicidad , Células A549 , Sustitución de Aminoácidos , Animales , Chlorocebus aethiops , Cromosomas Artificiales Bacterianos/genética , ADN Complementario/genética , Femenino , Humanos , Ratones , Ratones Noqueados , ARN Viral/genética , Receptor de Interferón alfa y beta/deficiencia , Receptor de Interferón alfa y beta/genética , Genética Inversa , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Células Vero , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología , Replicación Viral , Virus Zika/inmunología , Infección por el Virus Zika/prevención & controlRESUMEN
Deposition of the H2A.Z histone variant by the SWR1 complex (SWR1-C) in regulatory regions of specific loci modulates transcription. Characterization of mutations in Arabidopsis thaliana homologs of yeast SWR1-C has revealed a role for H2A.Z exchange in a variety of developmental processes. Nevertheless, the exact composition of plant SWR1-C and how it is recruited to target genes remains to be established. Here we show that SWC4, the Arabidopsis homolog of yeast SANT domain protein Swc4/Eaf2, is a DNA-binding protein that interacts with SWR1-C subunits. We demonstrate that the swc4-1 knockout mutant is embryo-lethal, while SWC4 RNAi knockdown lines display pleiotropic phenotypic alterations in vegetative and reproductive traits, including acceleration of flowering time, indicating that SWC4 controls post-embryonic processes. Transcriptomic analyses and genome-wide profiling of H2A.Z indicate that SWC4 represses transcription of a number of genes, including the floral integrator FT and key transcription factors, mainly by modulating H2A.Z deposition. Interestingly, SWC4 silencing does not affect H2A.Z deposition at the FLC locus nor expression of this gene, a master regulator of flowering previously shown to be controlled by SWR1-C. Importantly, we find that SWC4 recognizes specific AT-rich DNA elements in the chromatin regions of target genes and that SWC4 silencing impairs SWR1-C binding at FT. Collectively, our data suggest that SWC4 regulates plant growth and development by aiding SWR1-C recruitment and modulating H2A.Z deposition.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , ADN de Plantas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Histonas/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Unión al ADN/genética , Flores/crecimiento & desarrollo , Secuencia Rica en GC , Técnicas de Silenciamiento del Gen , Unión Proteica , Semillas/crecimiento & desarrolloRESUMEN
Fractional killing is the main cause of tumour resistance to chemotherapy. This phenomenon is observed even in genetically identical cancer cells in homogeneous microenvironments. To understand this variable resistance, here we investigate the individual responses to TRAIL in a clonal population of HeLa cells using live-cell microscopy and computational modelling. We show that the cellular mitochondrial content determines the apoptotic fate and modulates the time to death, cells with higher mitochondrial content are more prone to die. We find that all apoptotic protein levels are modulated by the mitochondrial content. Modelling the apoptotic network, we demonstrate that these correlations, and especially the differential control of anti- and pro-apoptotic protein pairs, confer mitochondria a powerful discriminatory capacity of apoptotic fate. We find a similar correlation between the mitochondria and apoptotic proteins in colon cancer biopsies. Our results reveal a different role of mitochondria in apoptosis as the global regulator of apoptotic protein expression.
Asunto(s)
Apoptosis/genética , Expresión Génica/genética , Mitocondrias/genética , Transducción de Señal/genética , Algoritmos , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Muerte Celular/genética , Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Mitocondrias/metabolismo , Modelos Genéticos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/farmacologíaRESUMEN
The large size of the coronavirus (CoV) genome (around 30 kb) and the instability in bacteria of plasmids carrying CoV replicase sequences represent serious restrictions for the development of CoV infectious clones using reverse genetic systems similar to those used for smaller positive sense RNA viruses. To overcome these problems, several approaches have been established in the last 13 years. Here we describe the engineering of CoV full-length cDNA clones as bacterial artificial chromosomes (BACs), using the Middle East respiratory syndrome CoV (MERS-CoV) as a model.
Asunto(s)
Cromosomas Artificiales Bacterianos/genética , Coronavirus/genética , ADN Complementario/genética , Animales , Línea Celular , Cricetinae , Escherichia coli , Ingeniería Genética , Humanos , Plásmidos/genética , Plásmidos/aislamiento & purificación , Genética Inversa , Transformación GenéticaRESUMEN
UNLABELLED: A 32-nucleotide (nt) RNA motif located at the 3' end of the transmissible gastroenteritis coronavirus (TGEV) genome was found to specifically interact with the host proteins glutamyl-prolyl-tRNA synthetase (EPRS) and arginyl-tRNA synthetase (RRS). This RNA motif has high homology in sequence and secondary structure with the gamma interferon-activated inhibitor of translation (GAIT) element, which is located at the 3' end of several mRNAs encoding proinflammatory proteins. The GAIT element is involved in the translation silencing of these mRNAs through its interaction with the GAIT complex (EPRS, heterogeneous nuclear ribonucleoprotein Q, ribosomal protein L13a, and glyceraldehyde 3-phosphate dehydrogenase) to favor the resolution of inflammation. Interestingly, we showed that the viral RNA motif bound the GAIT complex and inhibited the in vitro translation of a chimeric mRNA containing this RNA motif. To our knowledge, this is the first GAIT-like motif described in a positive RNA virus. To test the functional role of the GAIT-like RNA motif during TGEV infection, a recombinant coronavirus harboring mutations in this motif was engineered and characterized. Mutations of the GAIT-like RNA motif did not affect virus growth in cell cultures. However, an exacerbated innate immune response, mediated by the melanoma differentiation-associated gene 5 (MDA5) pathway, was observed in cells infected with the mutant virus compared with the response observed in cells infected with the parental virus. Furthermore, the mutant virus was more sensitive to beta interferon than the parental virus. All together, these data strongly suggested that the viral GAIT-like RNA motif modulates the host innate immune response. IMPORTANCE: The innate immune response is the first line of antiviral defense that culminates with the synthesis of interferon and proinflammatory cytokines to limit virus replication. Coronaviruses encode several proteins that interfere with the innate immune response at different levels, but to date, no viral RNA counteracting antiviral response has been described. In this work, we have characterized a 32-nt RNA motif located at the 3' end of the TGEV genome that specifically interacted with EPRS and RRS. This RNA motif presented high homology with the GAIT element, involved in the modulation of the inflammatory response. Moreover, the disruption of the viral GAIT-like RNA motif led to an exacerbated innate immune response triggered by MDA5, indicating that the GAIT-like RNA motif counteracts the host innate immune response. These novel findings may be of relevance for other coronaviruses and could serve as the basis for the development of novel antiviral strategies.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Arginino-ARNt Ligasa/metabolismo , Evasión Inmune , Inmunidad Innata , ARN Viral/metabolismo , Virus de la Gastroenteritis Transmisible/inmunología , Animales , Sitios de Unión , Línea Celular , Análisis Mutacional de ADN , Humanos , Unión Proteica , ARN Viral/genética , Virus de la Gastroenteritis Transmisible/genéticaRESUMEN
Coronaviruses (CoVs) infect humans and many animal species, and are associated with respiratory, enteric, hepatic, and central nervous system diseases. The large size of the CoV genome and the instability of some CoV replicase gene sequences during its propagation in bacteria, represent serious obstacles for the development of reverse genetic systems similar to those used for smaller positive sense RNA viruses. To overcome these limitations, several alternatives to more conventional plasmid-based approaches have been established in the last 13 years. In this report, we briefly review and discuss the different reverse genetic systems developed for CoVs, paying special attention to the severe acute respiratory syndrome CoV (SARS-CoV).
RESUMEN
Coronaviruses (CoVs) infect humans and many animal species, and are associated with respiratory, enteric, hepatic, and central nervous system diseases. The large size of the CoV genome and the instability of some CoV replicase gene sequences during its propagation in bacteria, represent serious obstacles for the development of reverse genetic systems similar to those used for smaller positive sense RNA viruses. To overcome these limitations, several alternatives to more conventional plasmid-based approaches have been established in the last 13 years. In this report, we briefly review and discuss the different reverse genetic systems developed for CoVs, paying special attention to the severe acute respiratory syndrome CoV (SARS-CoV).
Asunto(s)
Coronavirus/genética , Genética Inversa/métodos , Virología/métodos , Células Clonales , ReplicónRESUMEN
UNLABELLED: Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. Following transfection with the cDNA clone, infectious virus was rescued in both Vero A66 and Huh-7 cells. Recombinant MERS-CoVs (rMERS-CoVs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cDNA clones with these genes deleted. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. In contrast, an engineered mutant virus lacking the structural E protein (rMERS-CoV-ΔE) was not successfully rescued, since viral infectivity was lost at early passages. Interestingly, the rMERS-CoV-ΔE genome replicated after cDNA clone was transfected into cells. The infectious virus was rescued and propagated in cells expressing the E protein in trans, indicating that this virus was replication competent and propagation defective. Therefore, the rMERS-CoV-ΔE mutant virus is potentially a safe and promising vaccine candidate to prevent MERS-CoV infection. IMPORTANCE: Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans, whereas it would only survive a single virus infection cycle in vivo. This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection.
Asunto(s)
Infecciones por Coronaviridae/virología , Coronaviridae/fisiología , Vacunas Virales/genética , Replicación Viral , Línea Celular , Coronaviridae/genética , Coronaviridae/inmunología , Ingeniería Genética , Humanos , Proteínas Virales/genética , Proteínas Virales/inmunología , Vacunas Virales/inmunologíaRESUMEN
Coronavirus RNA synthesis is a sophisticated process performed by a viral multienzymatic replicase complex, together with cellular factors. A key enzyme of this replication complex is the RNA dependent RNA polymerase (RdRp). To study the replication of coronavirus genome, six monoclonal antibodies (mAbs) specific for transmissible gastroenteritis virus (TGEV) RdRp were generated and characterized. His-tagged RdRp was expressed in baculovirus, purified and used as immunogen to produce mAbs. The TGEV RdRp was recognized by these mAbs in the context of virus infection by immunofluorescence analysis and Western blot. Epitope mapping by Pepscan indicated that RdRp mAbs recognized four non-overlapping linear epitopes located in a 62-amino acid region of the N-terminal domain, suggesting that this region may constitute an immunodominant domain. The availability of TGEV RdRp mAbs will be instrumental to study coronavirus replication and to analyze the function of RdRp in pathogenesis.
Asunto(s)
Mapeo Epitopo/métodos , ARN Polimerasa Dependiente del ARN/análisis , ARN Polimerasa Dependiente del ARN/inmunología , Virus de la Gastroenteritis Transmisible/química , Virus de la Gastroenteritis Transmisible/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Antígenos Virales/inmunología , Baculoviridae/genética , Western Blotting , Línea Celular , Coronavirus/genética , Coronavirus/inmunología , Epítopos/genética , Epítopos/inmunología , Técnica del Anticuerpo Fluorescente , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Porcinos , Virus de la Gastroenteritis Transmisible/genéticaRESUMEN
The ubiquitin pathway is emerging as a powerful system that controls the stability of key regulatory proteins. In plants, this pathway plays an important role in controlling several developmental processes, responses to environmental changes and also cell division. Arabidopsis SKP2A is an F-box protein that regulates the stability of the E2FC-DPB transcription factor, a repressor of cell proliferation. Although the function of SKP2A is to recruit targets for degradation, we have shown that SKP2A is also degraded through the Ub/26S pathway and, interestingly, auxin stimulates such degradation. Overexpression of SKP2A positively regulates cell division, increasing the number of cells in G(2)/M, reducing the level of ploidy and developing higher number of lateral root primordia. In addition, we showed in this report that overexpression of SKP2A increased the survival of Arabidopsis plants when they grown on a medium with high levels of sucrose, likely by maintaining cell division active. Thus, it is likely that SKP2A connects cell division with stress responses.