Identificación y caracterización de un motivo de RNA similar al elemento GAIT en el extremo 3' del genoma del TGEV que modula la respuesta inmune innata

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 30-04-2015Coronaviruses (CoVs) are enveloped, positive-sense RNA viruses that belong to the order Nidovirales, causing respiratory and enteric infections in a wide range of animals and human. CoV replication and transcription take place at cytoplasmic double membrane vesicles and is mediated by the viral replicase. These processes require the specific recognition of RNA cis-acting signals located at the ends of the viral genome. Similarly to many other RNA viruses, the virus-encoded replication complex presumably associates with host-cell proteins to complete the synthesis of viral RNA. Using transmissible gastroenteritis coronavirus (TGEV) as a model, we previously identified nine cellular proteins interacting with the genome 3’ end, including the heterogeneous nuclear ribonucleoproteins (hnRNPs) A0, A1, A2/B1, Q and U, the translational factors glutamyl-prolyl-tRNA synthetase (EPRS), arginyl-tRNA synthetase (RRS) and poly(A)-binding protein (PABP), and the p100 transcriptional coactivator. From these proteins, a functional role on viral RNA synthesis was reported for hnRNP Q, EPRS and PABP. In this thesis, the functional study was extended to the proteins RRS and p100, and a positive role of both cell proteins in the viral RNA synthesis was demonstrated by silencing analysis. The RNA domains interacting with the cell proteins involved in TGEV RNA synthesis were further analyzed to study their mechanisms of action. After several RNA mapping stages, a 32-nt RNA motif located at the 3’ end of the TGEV genome was found to specifically interact with EPRS and RRS. This interaction was also observed during the infection, where both tRNA synthetases specifically interacted with the viral genomic and subgenomic RNAs. Interestingly, both aminoacil tRNA synthetases were incorporated into the viral particle, possibly through their interaction with the viral genome RNA. This RNA motif presented high homology in sequence and secondary structure with the gamma interferon activated inhibitor of translation (GAIT) element, which is present at the 3’ end of several mRNAs coding proinflammatory proteins. The GAIT element is involved in the translation silencing of these mRNAs through its interaction with the GAIT complex [EPRS, hnRNP Q, L13a, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] to favor the resolution of inflammation. Similarly to the cellular GAIT element, the viral RNA domain was able to bind the GAIT complex and inhibit the in vitro translation of a chimeric mRNA containing this motif, suggesting that the viral RNA domain could constitute the first GAIT-like motif described in a positive RNA virus. To test the functional role of the GAIT-like motif in TGEV infection, two recombinant viruses harboring mutations in this motif were engineered and characterized. Abrogation of the GAIT-like 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 viruses compared with the parental virus infection. Furthermore, mutant viruses were more sensitive to interferon beta than the parental virus. Altogether, these data suggested that the GAIT-like motif modulates the host innate immune response

Engineering Infectious cDNAs of Coronavirus as Bacterial Artificial Chromosomes

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 thirteen 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.This work was supported by grants from the Ministry of Science and Innovation of Spain (MCINN) (BIO2010-16705), the European Community's Seventh Framework Programme (FP7/2007-2013) under the project “EMPERIE” (HEALTH-F3-2009-223498), and the National Institute of Health (NIH) of USA (2P01AI060699-06A1). S. M. received a predoctoral fellowship from the National Institute of Health (ISCIII) of Spain

Identification of a gamma interferon-activated inhibitor of translation-like RNA motif at the 3′ end of the transmissible gastroenteritis coronavirus genome modulating innate immune response

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.This work was supported by grants from the Ministry of Science and Innovation of Spain (MCINN) (BIO2010-16705), the European Community’s Seventh Framework Programme (FP7/2007-2013) under the project “EMPERIE” (HEALTH-F3-2009-223498), and the National Institutes of Health (NIH) of The United States (2P01AI060699-06A1). S.M.-J. received a predoctoral fellowship from the National Institutes of Health (ISCIII) of Spain and a contract from the National Institutes of Health (NIH) of The United States. A.N. and S.Z. received contracts from EU and NI

An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo

The recent outbreaks of Zika virus (ZIKV), its association with Guillain–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.This research was funded by the Spanish Ministry of Economy and Competitiveness (MINECO) (grant number BFU2016-79127-R) to F.A. and F.J.I. and the National Institute of Health (NIH) (grant number 1R21AI120500) to L.M.-S. and F.A.Peer reviewe

Immunogenic characterization and epitope mapping of transmissible gastroenteritis virus RNA dependent RNA polymerase

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.This work was supported by grants from the Ministry of Science and Innovation of Spain (MCINN) (BIO2010-16705), the Spanish National Research Council (CSIC) (projects 200920I024, 200920I016 and 201120E007), the Community of Madrid (S-SAL-0185-2006), the European Community's Seventh Framework Programme (FP7/2007-2013) under the projects “PLAPROVA” (KBBE-227056) and “EMPERIE” (223498), and Pfizer Animal Health. AN and SM received a predoctoral fellowship from MCINN and from National Institute of Health (ISCIII) of Spain, respectively. CG and MG received a contract from the Spanish National Research Council (CSIC)

Reprint of: Coronavirus reverse genetic systems: Infectious clones and replicons

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).This work was supported by grants from the Ministry of Science and Innovation of Spain (MCINN) (BIO2010-16705), the European Community's Seventh Framework Programme (FP7/2007–2013) under the project “EMPERIE” (HEALTH-F3-2009-223498), and the National Institute of Health (NIH) of USA (2P01AI060699-06A1)

Coronavirus reverse genetic systems: Infectious clones and replicons

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).This work was supported by grants from the Ministry of Science and Innovation of Spain (MCINN) (BIO2010-16705), the European Community’s Seventh Framework Programme (FP7/2007–2013) under the project “EMPERIE” (HEALTH-F3-2009-223498), and the National Institute of Health (NIH) of USA (2P01AI060699-06A1)

Mitochondrial levels determine variability in cell death by modulating apoptotic gene expression

It is unclear what causes variation in cell death in response to chemotherapy. Here, the authors show that cellular mitochondrial content modulates apoptotic protein levels, which in turn regulates response to agents such as TRAIL

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