Mecanismos de entrada y de organización del complejo de replicación del virus de la fiebre aftosa: estudio comparativo con el virus de la enfermedad vesicular del cerdo y el virus de la estomatitits vesicular

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 29-05-0

Reconciling west nile virus with the autophagic pathway

© 2015, Taylor & Francis Group, LLC. West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus responsible for recurrent outbreaks of meningitis and encephalitis. Several studies analyzing the interactions of this pathogen with the autophagic pathway have reported opposite results with evidence for and against the upregulation of autophagy in infected cells. In this regard, we have recently reported that minimal genetic changes (single amino acid substitutions) in nonstructural proteins of WNV can modify the ability of the virus to induce autophagic features such as LC3 modification and aggregation in infected cells. We think that these results could help explain some of the previously reported discrepancies. These findings could also aid in deciphering the interactions of this pathogen with the autophagic pathway at the molecular level aimed to develop feasible antiviral strategies to combat this pathogen, and other related flaviviruses.INIA (RTA2011-0036 and E-RTA2013-00013-C01) and the Comunidad Autónoma de Madrid PLATESA (P2013/ABI-2906)Peer Reviewe

Plasma Membrane Phosphatidylinositol 4,5 Bisphosphate Is Required for Internalization of Foot-and-Mouth Disease Virus and Vesicular Stomatitis Virus

Phosphatidylinositol-4,5-bisphosphate, PI(4,5)P 2, is a phospholipid which plays important roles in clathrin-mediated endocytosis. To investigate the possible role of this lipid on viral entry, two viruses important for animal health were selected: the enveloped vesicular stomatitis virus (VSV) - which uses a well characterized clathrin mediated endocytic route - and two different variants of the non-enveloped foot-and-mouth disease virus (FMDV) with distinct receptor specificities. The expression of a dominant negative dynamin, a PI(4,5)P 2 effector protein, inhibited the internalization and infection of VSV and both FMDV isolates. Depletion of PI(4,5)P 2 from plasma membrane using ionomycin or an inducible system, and inhibition of its de novo synthesis with 1-butanol revealed that VSV as well as FMDV C-S8c1, which uses integrins as receptor, displayed a high dependence on PI(4,5)P 2 for internalization. Expression of a kinase dead mutant (KD) of phosphatidylinositol-4-phosphate-5-kinase Iα (PIP5K-Iα), an enzyme responsible for PI(4,5)P 2 synthesis that regulates clathrin-dependent endocytosis, also impaired entry and infection of VSV and FMDV C-S8c1. Interestingly FMDV MARLS variant that uses receptors other than integrins for cell entry was less sensitive to PI(4,5)P 2 depletion, and was not inhibited by the expression of the KD PIP5K-Iα mutant suggesting the involvement of endocytic routes other than the clathrin-mediated on its entry. These results highlight the role of PI(4,5)P 2 and PIP5K-Iα on clathrin-mediated viral entry. © 2012 Vázquez-Calvo et al.BIO2008-0447-C03-01 and BIO2011-24351; Fundación Ramón ArecesPeer Reviewe

Synapse Loss in Olfactory Local Interneurons Modifies Perception

Synapse loss correlates with cognitive decline in aging and most neurological pathologies. Sensory perception changes often represent subtle dysfunctions that precede the onset of a neurodegenerative disease. However, a cause–effect relationship between synapse loss and sensory perception deficits is difficult to prove and quantify due to functional and structural adaptation of neural systems. Here we modified a PI3K/AKT/GSK3 signaling pathway to reduce the number of synapses—without affecting the number of cells—in five subsets of local interneurons of the Drosophila olfactory glomeruli and measured the behavioral effects on olfactory perception. The neuron subsets were chosen under the criteria of GABA or ChAT expression. The reduction of one subset of synapses, mostly inhibitory, converted the responses to all odorants and concentrations tested as repulsive, while the reduction of another subset, mostly excitatory, led to a shift toward attraction. However, the simultaneous reduction of both synapse subsets restored normal perception. One group of local interneurons proved unaffected by the induced synapse loss in the perception of some odorants, indicating a functional specialization of these cells. Using genetic tools for space and temporal control of synapse number decrease, we show that the perception effects are specific to the local interneurons, rather than the mushroom bodies, and are not based on major structural changes elicited during development. These findings demonstrate that synapse loss cause sensory perception changes and suggest that normal perception is based on a balance between excitation and inhibition.Peer reviewe

A single amino acid substitution in the capsid of foot-and-mouth disease virus can increase acid resistance

Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their disassembly at pH values slightly below neutrality. This acid-dependent disassembly process is required for viral RNA release inside endosomes. To study the molecular determinants of viral resistance to acid-induced disassembly, six FMDV variants with increased resistance to acid inactivation were isolated. Infection by these mutants was more sensitive to drugs that raise the endosomal pH (NH4Cl and concanamycin A) than was infection by the parental C-S8c1 virus, confirming that the increase in acid resistance is related to a lower pH requirement for productive uncoating. Amino acid replacement N17D at the N terminus of VP1 capsid protein was found in all six mutants. This single substitution was shown to be responsible for increased acid resistance when introduced into an infectious FMDV clone. The increased resistance of this mutant against acid-induced inactivation was shown to be due to its increased resistance against capsid dissociation into pentameric subunits. Interestingly, the N17D mutation was located close to but not at the interpentamer interfaces. The mutants described here extend the panel of FMDV variants exhibiting different pH sensitivities and illustrate the adaptive flexibility of viral quasispecies to pH variations.Work at the F.S. laboratory was supported by grants from Ministerio de Ciencia e Innovación (MICINN) BIO2008-0447-C03-01 and CSD2006-0007; work at the M.G.M. laboratory was supported by grants from MICINN (BIO2009-10092) and Comunidad de Madrid (S-2009/MAT/1467). An institutional grant from Fundación Ramón Areces is also acknowledged.Peer Reviewe

Limited susceptibility of mice to Usutu virus (USUV) infection and induction of flavivirus cross-protective immunity

© 2015 Elsevier Inc. Flaviviruses are RNA viruses that constitute a worrisome threat to global human and animal health. In Europe, West Nile virus (WNV) outbreaks have dramatically increased in number and severity in recent years, with dozens of human and horse deaths and a high avian mortality across the continent. Besides WNV, the only clinically relevant mosquito-borne flavivirus detected so far in Europe has been the Usutu virus (USUV), which after being reported for the first time in Austria in 2001, quickly spread across Europe, causing a considerable number of bird deaths and neurological disorders in a few immunocompromised patients. Even though USUV infects multiple avian species that develop antibodies, there is little information about USUV susceptibility, pathogenicity and cross-reactive immunity. Here, the susceptibility of suckling and adult mice to USUV infection and the induction of cross-protective immunity against WNV challenge have been addressed.Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), PLATESA (P2013/ABI-2906) from the Comunidad Autónoma de Madrid, and by the Network of Animal Disease Infectiology and Research-European Union (NADIR-EU- 228394)Peer Reviewe

Modification of the host cell lipid metabolism induced by hypolipidemic drugs targeting the acetyl coenzyme A carboxylase impairs West Nile virus replication

West Nile virus (WNV) is a neurotropic flavivirus transmitted by the bite of mosquitoes that causes meningitis and encephalitis in humans, horses, and birds. Several studies have highlighted that flavivirus infection is highly dependent on cellular lipids for virus replication and infectious particle biogenesis. The first steps of lipid synthesis involve the carboxylation of acetyl coenzyme A (acetyl-CoA) to malonyl-CoA that is catalyzed by the acetyl-CoA carboxylase (ACC). This makes ACC a key enzyme of lipid synthesis that is currently being evaluated as a therapeutic target for different disorders, including cancers, obesity, diabetes, and viral infections. We have analyzed the effect of the ACC inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA) on infection by WNV. Lipidomic analysis of TOFA-treated cells confirmed that this drug reduced the cellular content of multiple lipids, including those directly implicated in the flavivirus life cycle (glycerophospholipids, sphingolipids, and cholesterol). Treatment with TOFA significantly inhibited the multiplication of WNV in a dose-dependent manner. Further analysis of the antiviral effect of this drug showed that the inhibitory effect was related to a reduction of viral replication. Furthermore, treatment with another ACC inhibitor, 3,3,14,14-tetramethylhexadecanedioic acid (MEDICA 16), also inhibited WNV infection. Interestingly, TOFA and MEDICA 16 also reduced the multiplication of Usutu virus (USUV), a WNV-related flavivirus. These results point to the ACC as a druggable cellular target suitable for antiviral development against WNV and other flaviviruses.BIO2011-24351 (to F.S.), AGL2014-52395-C2-1-R (to F.S.), E-RTA2013-0013 (to J.-C.S. and F.S.), S2013/ABI-2906 (to J.-C.S. and F.S.), AGL2014-56518-JIN (to M.A.M.-A.), and CTQ2014-54743-R (to J.C.). Work at CBMSO was also supported by Fundación Ramón Areces.Peer Reviewe

Nanobodies Protecting From Lethal SARS-CoV-2 Infection Target Receptor Binding Epitopes Preserved in Virus Variants Other Than Omicron

The emergence of SARS-CoV-2 variants that escape from immune neutralization are challenging vaccines and antibodies developed to stop the COVID-19 pandemic. Thus, it is important to establish therapeutics directed toward multiple or specific SARS-CoV-2 variants. The envelope spike (S) glycoprotein of SARS-CoV-2 is the key target of neutralizing antibodies (Abs). We selected a panel of nine nanobodies (Nbs) from dromedary camels immunized with the receptor-binding domain (RBD) of the S, and engineered Nb fusions as humanized heavy chain Abs (hcAbs). Nbs and derived hcAbs bound with subnanomolar or picomolar affinities to the S and its RBD, and S-binding cross-competition clustered them in two different groups. Most of the hcAbs hindered RBD binding to its human ACE2 (hACE2) receptor, blocked cell entry of viruses pseudotyped with the S protein and neutralized SARS-CoV-2 infection in cell cultures. Four potent neutralizing hcAbs prevented the progression to lethal SARS-CoV-2 infection in hACE2-transgenic mice, demonstrating their therapeutic potential. Cryo-electron microscopy identified Nb binding epitopes in and out the receptor binding motif (RBM), and showed different ways to prevent virus binding to its cell entry receptor. The Nb binding modes were consistent with its recognition of SARS-CoV-2 RBD variants; mono and bispecific hcAbs efficiently bound all variants of concern except omicron, which emphasized the immune escape capacity of this latest variant.This work was partially funded by Ministerio de Ciencia e Innovación (MICIN; https://www.ciencia.gob.es/) and the Spanish Research Council (CSIC; https://www.csic.es/) under grants PIE-RD-COVID 19 (No 202020E079) and PTI+ Salud Global REC_EU (No SGL 2103051, NextGenerationEU) to LF, JMC, PG, and UG, and (No SGL 2103053, NextGenerationEU) to MM-A. This study was partially conducted within the CSIC Antiviral Screening Network, an infrastructure supported by NextGeneration EU funds (https://ec.europa.eu/info/strategy/recovery-plan-europe_es) from the European Union and the European Virus Archive Global (EVag) of the European Union’s Horizon 2020 (https://ec.europa.eu/programmes/horizon2020/en/home) research and innovation programme (No 871029) to PG and UG. EM facilities of CNB-CSIC were supported by Ministerio de Ciencia e Innovación (MICIN; https://www.ciencia.gob.es/), EU-FEDER (https://ec.europa.eu/regional_policy/es/funding/erdf/) CRIOMECORR project (ESFRI-2019-01-CSIC-16). JMC access to the European Synchrotron Radiation Facility (ESRF) CM01 line through the Iberian-BAG, and to the Instruct Image Processing Center (I2PC, http://i2pc.es/) by projects PID16168 and PID14989. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Innovación en las enseñanzas universitarias: experiencias presentadas en las III Jornadas de Innovación Educativa de la ULL

En este libro se recoge un conjunto de experiencias de innovación educativa desarrolladas en la ULL en el curso 2011-12. Se abordan distintos ámbitos y ramas del conocimiento, y ocupan temáticas variadas que han sido desarrolladas con rigor, y con un claro potencial para su extrapolación a efectos de la mejora educativa en el ámbito universitario. Esta publicación constituye una primera edición de una serie que irá recogiendo las experiencias de innovación educativa de la ULL. Este es un paso relevante para su impulso en nuestra institución, como lo es el de su vinculación con la investigación educativa, para potenciar su publicación en las revistas científicas en este ámbito cada vez más pujante y relevante para las universidades. Sobre todo representan el deseo y el compromiso del profesorado de la ULL para la mejora del proceso educativo mediante la investigación, la evaluación y la reflexión compartida de nuestras prácticas y planteamientos docentes