Structural and Functional Analysis of Dengue Virus RNA

Sequences and structures present at the 5' and 3' UTRs of RNA viruses play crucial roles in the initiation and regulation of translation, RNA synthesis and viral assembly. In dengue virus, as well as in other mosquito-borne flaviviruses, the presence of complementary sequences at the ends of the genome mediate long-range RNA-RNA interactions. Dengue virus RNA displays two pairs of complementary sequences (CS and UAR) required for genome circularization and viral viability. In order to study the molecular mechanism by which these RNA-RNA interactions participate in the viral life cycle, we developed a dengue virus replicon system. RNA transfection of the replicon in mosquito and mammalian cells allows discrimination between RNA elements involved in translation and RNA synthesis. We found that mutations within CS or UAR at the 5' or 3' ends of the RNA that interfere with base pairing did not significantly affect translation of the input RNA but seriously compromised or abolished RNA synthesis. Furthermore, a systematic mutational analysis of UAR sequences indicated that, beside the role in RNA cyclization, specific nucleotides within UAR are also important for efficient RNA synthesis.Fil: Alvarez, Diego Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Lodeiro, María Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Filomatori, Claudia Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Fucito, Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Mondotte, Juan Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Gamarnik, Andrea Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

A Long-Chain Flavodoxin Protects Pseudomonas aeruginosa from Oxidative Stress and Host Bacterial Clearance

Long-chain flavodoxins, ubiquitous electron shuttles containing flavin mononucleotide (FMN) as prosthetic group, play an important protective role against reactive oxygen species (ROS) in various microorganisms. Pseudomonas aeruginosa is an opportunistic pathogen which frequently has to face ROS toxicity in the environment as well as within the host. We identified a single ORF, hereafter referred to as fldP (for flavodoxin from P. aeruginosa), displaying the highest similarity in length, sequence identity and predicted secondary structure with typical long-chain flavodoxins. The gene was cloned and expressed in Escherichia coli. The recombinant product (FldP) could bind FMN and exhibited flavodoxin activity in vitro. Expression of fldP in P. aeruginosa was induced by oxidative stress conditions through an OxyR-independent mechanism, and an fldP-null mutant accumulated higher intracellular ROS levels and exhibited decreased tolerance to H2O2 toxicity compared to wild-type siblings. The mutant phenotype could be complemented by expression of a cyanobacterial flavodoxin. Overexpression of FldP in a mutT-deficient P. aeruginosa strain decreased H2O2-induced cell death and the hypermutability caused by DNA oxidative damage. FldP contributed to the survival of P. aeruginosa within cultured mammalian macrophages and in infected Drosophila melanogaster, which led in turn to accelerated death of the flies. Interestingly, the fldP gene is present in some but not all P. aeruginosa strains, constituting a component of the P. aeruginosa accessory genome. It is located in a genomic island as part of a self-regulated polycistronic operon containing a suite of stress-associated genes. The collected results indicate that the fldP gene encodes a long-chain flavodoxin, which protects the cell from oxidative stress, thereby expanding the capabilities of P. aeruginosa to thrive in hostile environments.Fil: Moyano, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Química Biológica de Córdoba (p); ArgentinaFil: Tobares, Romina Alín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Química Biológica de Córdoba (p); ArgentinaFil: Rizzi, Yanina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Química Biológica de Córdoba (p); ArgentinaFil: Krapp, Adriana del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Mondotte, Juan Alberto. Instituto Pasteur; FranciaFil: Bocco, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Bioquímica Clínica E Inmunología; ArgentinaFil: Saleh, Maria Carla. Instituto Pasteur; FranciaFil: Carrillo, Nestor Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Smania, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Química Biológica de Córdoba (p); Argentin

An analogue of the antibiotic teicoplanin prevents flavivirus entry in vitro.

There is an urgent need for potent inhibitors of dengue virus (DENV) replication for the treatment and/or prophylaxis of infections with this virus. We here report on an aglycon analogue of the antibiotic teicoplanin (code name LCTA-949) that inhibits DENV-induced cytopathic effect (CPE) in a dose-dependent manner. Virus infection was completely inhibited at concentrations that had no adverse effect on the host cells. These findings were corroborated by quantification of viral RNA levels in culture supernatant. Antiviral activity was also observed against other flaviviruses such as the yellow fever virus and the tick-borne encephalitis virus (TBEV). In particular, potent antiviral activity was observed against TBEV. Time-of-drug-addition experiments indicated that LCTA-949 inhibits an early stage in the DENV replication cycle; however, a virucidal effect was excluded. This observation was corroborated by the fact that LCTA-949 lacks activity on DENV subgenomic replicon (that does not encode structural proteins) replication. Using a microsopy-based binding and fusion assay employing DiD-labeled viruses, it was shown that LCTA-949 targets the early stage (binding/entry) of the infection. Moreover, LCTA-949 efficiently inhibits infectivity of DENV particles pre-opsonized with antibodies, thus potentially also inhibiting antibody-dependent enhancement (ADE). In conclusion, LCTA-949 exerts in vitro activity against several flaviviruses and does so (as shown for DENV) by interfering with an early step in the viral replication cycle.Fil: De Burghgraeve, Tine. Katholikie Universiteit Leuven; BélgicaFil: Kaptein, Suzanne J. F.. Katholikie Universiteit Leuven; BélgicaFil: Ayala Nunez, Nilda V.. University of Groningen; Países BajosFil: Mondotte, Juan Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Pastorino, Boris. Université de la Méditerranée; FranciaFil: Printsevskaya, Svetlana S.. Russian Academy of Medical Sciences; RusiaFil: de Lamballerie, Xavier. Université de la Méditerranée; FranciaFil: Jacobs, Michael. Royal Free & University College Medical School; Reino UnidoFil: Preobrazhenskaya, Maria. Russian Academy of Medical Sciences; RusiaFil: Gamarnik, Andrea Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Smit, Jolanda M.. University of Groningen; Países BajosFil: Neyts, Johan. Katholikie Universiteit Leuven; Bélgic

Interactions between dengue virus and the host cell

El virus del dengue produce en humanos la enfermedad viral más frecuentemente transmitida por artrópodos y no existe hasta el momento terapia antiviral ni vacuna alguna. En este trabajo se desarrollaron diferentes herramientas genéticas con el fin de estudiar las distintas etapas de la replicación viral y la interacción del virus con la célula huésped. Así, se obtuvo el primer clon infeccioso del virus del dengue de un aislamiento argentino y distintas generaciones de virus con proteínas reporteras. Empleando estos sistemas genéticos se pudieron disecar las distintas etapas de la replicación viral. La manipulación del genoma viral permitió caracterizar mutantes de N-glicosilación de las proteínas de envoltura y pre-membrana, y estudiar su importancia en las distintas etapas del ciclo viral. El ensayo del virus reportero, el cual permite evaluar la actividad de luciferasa como un marcador de replicación viral, fue miniaturizado para realizar una búsqueda de quinasas de la célula huésped involucradas en la replicación. Para esto se utilizó la tecnología del RNA de interferencia a gran escala. Mediante este ensayo se identificaron distintas quinasas claves en la replicación del virus y se estudió su importancia en la replicación viral. Por último, se aportó nueva infomación sobre el mecanismo celular por el cual la proteína de cápside se dirige a las organelas celulares lipid droplets, y su relación con la proteína celular ADRP. Este trabajo provee nuevas herramientas genéticas y nueva información sobre los requerimientos del virus del dengue para replicar en la célula infectada. Además, realiza un aporte que podría llevar al desarrollo de nuevas estrategias antivirales.Dengue is the most prevalent viral disease transmitted by arthropods. At present, neither antiviral therapies or licensed vaccine exist. In this work, we developed new genetic tools to study different steps of viral replication and host-viral interactions. We obtained the first infectious clone from an Argentinean clinical isolate, and several dengue virus reporter systems. Using these tools, we dissected each step of the viral replication process through a luciferase activity assay. Genetically manipulating the viral genome, we characterized N-glycosylation mutants of envelope and pre-membrane proteins, and their role during the viral life cycle. The reporter virus assay was miniaturized to perform an RNA interference screen to search for host kinases involved in viral replication. We identified several kinases important for different steps of viral replication. Finally, we provided new information about the cellular mechanism by which the capsid protein is targeted to cellular organelles known as lipid droplets, and investigated the relationship of capsid with the cellular protein ADRP. This thesis provides new genetic tools and new information on the cellular requirements for dengue virus replication, which could lead to the development of new antiviral strategies.Fil:Mondotte, Juan Alberto. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Uncoupling cis-Acting RNA elements from coding sequences revealed a requirement of the N-terminal region of dengue virus capsid protein in virus particle formation

Little is known about the mechanism of flavivirus genome encapsidation. Here, functional elements of the dengue virus (DENV) capsid (C) protein were investigated. Study of the N-terminal region of DENV C has been limited by the presence of overlapping cis-acting RNA elements within the protein-coding region. To dissociate these two functions, we used a recombinant DENV RNA with a duplication of essential RNA structures outside the C coding sequence. By the use of this system, the highly conserved amino acids FNML, which are encoded in the RNA cyclization sequence 5'CS, were found to be dispensable for C function. In contrast, deletion of the N-terminal 18 amino acids of C impaired DENV particle formation. Two clusters of basic residues (R5-K6-K7-R9 and K17-R18-R20-R22) were identified as important. A systematic mutational analysis indicated that a high density of positive charges, rather than particular residues at specific positions, was necessary. Furthermore, a differential requirement of N-terminal sequences of C for viral particle assembly was observed in mosquito and human cells. While no viral particles were observed in human cells with a virus lacking the first 18 residues of C, DENV propagation was detected in mosquito cells, although to a level about 50-fold less than that observed for a wild-type (WT) virus. We conclude that basic residues at the N terminus of C are necessary for efficient particle formation in mosquito cells but that they are crucial for propagation in human cells. This is the first report demonstrating that the N terminus of C plays a role in DENV particle formation. In addition, our results suggest that this function of C is differentially modulated in different host cellsFil: Samsa, Marcelo Mario Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Mondotte, Juan Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Caramelo, Julio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Gamarnik, Andrea Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentin

Dengue Virus Uses a Non-Canonical Function of the Host GBF1-Arf-COPI System for Capsid Protein Accumulation on Lipid Droplets

Dengue viruses cause the most important human viral disease transmittedby mosquitoes. In recent years, a great deal has been learnedabout molecular details of dengue virus genome replication; however,little is known about genome encapsidation and the functions of theviral capsid protein. During infection, dengue virus capsid progressivelyaccumulates around lipid droplets (LDs) by an unknown mechanism.Here, we examined the process by which the viral capsid is transportedfrom the endoplasmic reticulum (ER) membrane, where the protein issynthesized, to LDs. Using different methods of intervention, we foundthat the GBF1-Arf1/Arf4-COPI pathway is necessary for capsid transportto LDs, while the process is independent of both COPII componentsand Golgi integrity. The transport was sensitive to Brefeldin A, while adrug resistant form of GBF1 was sufficient to restore capsid subcellulardistribution in infected cells. The mechanism by which LDs gain or loseproteins is still an open question. Our results support a model in whichthe virus uses a non-canonical function of the COPI system for capsidaccumulation on LDs, providing new ideas for antiviral strategies.Fil: Iglesias, Nestor Gabriel. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mondotte, Juan Alberto. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Byk, Laura Andrea. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: de Maio, Federico Andres. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Samsa, Marcelo Mario Alejandro. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alvarez, Cecilia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Bioquímica Clínica E Inmunología; ArgentinaFil: Gamarnik, Andrea Vanesa. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin