03B Virology and Microbiology

Virology and Microbiology
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Viral modulation of the immune response Antonio Alcami Pertejo Molecular bases of viral pathogenesis and anti-cancer potential Jos M. Almendral del Ro Molecular Ecology of Extreme Environments Ricardo Amils Pibernat Bacterial Cell Division and Antibiotics Resistance Juan Alfonso Ayala Serrano Biotechnology and Genetics of Extreme thermophilic Bacteria Jos Berenguer Carlos ASFV: virus models of evasion and protection ngel L. Lpez Carrascosa Bacterial Morphogenesis Miguel ngel de Pedro Montalbn Generic variability of RNA viruses Esteban Domingo Solans Gene expresin in Streptomyces and yeasts Antonio Jimnez / Mara Fernndez Lobato Virus Engineering Mauricio Garca Mateu Effects of extrachromosomal elements on behaviour of its host Bacillus Wilfried J.J. Meijer Human immunodeficiency virus reverse transcriptase and antiretroviral therapy Lus Menndez Arias African swine fever virus Maria Luisa Salas Falgueras New strategies for prevention and control of viral diseases: foot-and-mouth disease virus as a model Francisco Sobrino
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Viral modulation of the immune response

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Lnea Investigacin Staff Publications Other Activities
We are investigating immune evasion mechanisms employed by large DNA viruses, poxviruses and herpesviruses. Specifically, we are characterizing a unique immunomodulatory strategy that consists of the secretion of viral proteins that bind cytokines and chemokines, important mediators of the inflammatory and immune response. We work on two virus systems: (1) Herpesviruses like herpes simplex virus and human cytomegalovirus, which are human pathogens of clinical relevance; and (2) Poxviruses such as vaccinia virus, the smallpox vaccine, and variola virus, the causative agent of smallpox and the only viral disease eradicated as a result of a global vaccination campaign. Secreted cytokine decoy receptors are likely to contribute to the pathogenesis of variola virus, which was one of the most virulent viruses known by mankind. The immune modulatory activity of the viral cytokine receptors and their contribution to pathology are being addressed in the mousepox model. Mousepox is a smallpox-like disease caused by ectromelia virus, a natural mouse pathogen, and a classical model of viral pathogenesis. Viruses have optimized during millions of years of evolution their ability to manipulate the host immune response. Viruses offer a unique opportunity to develop their immune evasion strategies as novel therapeutic approaches to block the damage caused by an uncontrolled inflammatory response. In collaboration with Biotech Companies, we are working towards the development of viral immunomodulatory proteins as potential medicaments to treat human allergic and autoimmune diseases caused by immunopathological reactions. Viruses are the most abundant and diverse biological entities on Earth. We are interested in the molecular characterization of complex viral communities using new massive sequencing methodologies (Roche-454 Life Sciences). We have described for the first time the viral community in an Antarctic lake and are expanding these studies to other lakes along the Antarctic Peninsula and in the Arctic. We are also interested in using the new sequencing technologies to identify viruses associated with human pathologies, such as multiple sclerosis.
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Figure 1. Mimicry of cytokines and cytokine receptors by herpesviruses and poxviruses.
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Figure 2. Metagenomic studies of the viral community in Antarctic lakes.
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Home Group Leader: Antonio Alcami Pertejo Postdoctoral Fellows: Ali Alejo Herberg Abel Viejo Borbolla Alberto Lpez Bueno Daniel Rubio Muoz Elena Merino Rodrguez Soledad Blanco Chapinal Imma Montanuy Sellart Juan Alonso Lobo Graduate Students: Marcos Palomo Otero Sergio Martn Pontejo Nadia Martnez Martn Carla Mavin Haleh Heidarieh Technical Assistance: Roco Martn Hernndez Carolina Snchez Fernndez Visiting Scientists: Joanne Devlin (University of Melbourne, Australia) Julia Fahel (Trinity College, Dublin, Ireland)
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Andrs G., Leali, D., Mitola, S., Coltrini, D., Camozzi, M., Corsini, M., Belleri, M., Hirsch, E., Schwendener, R. A., Christofori, Alcami, A. and Presta, M. (2009) A pro-inflammatory signature mediates FGF2-induced angiogenesis. J. Cell. Mol. Med. 13, 2083-2108. Waibler, Z., Anzaghe, M., Frenz, T., Schwantes, A., Phlmann, C., Ludwig, H., Palomo-Otero, M., Alcami, A., Sutter, G. and Kalinke, U. (2009) Vaccinia virus-mediated inhibition of type I interferon responses is a multi-factorial process involving the soluble type I interferon receptor B18 and intracellular components. J. Virol. 83, 1563-1570.
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Carson, C., Antoniou, M., Ruiz-Argello, M. B., Alcami, A., Christodoulou, V., Messaritakis, I., Blackwell, J. M. and Courtenay, O. (2009) A prime/boost DNA/Modified vaccinia virus Ankara vaccine expressing recombinant Leishmania DNA encoding TRYP is safe and immunogenic in outbred dogs, the reservoir of zoonotic visceral leishmaniasis. Vaccine 27, 1080-1086. Alcami, A. and Saraiva, M. (2009) Chemokine binding proteins encoded by pathogens. Adv. Exp. Med. Biol. 666, 167-179. Poole, E., Groves, I., Ho, Y., Benedict, C., Alcami, A. and Sinclair, J. (2009) Identification of TRIM23 as a co-factor involved in the regulation of NFkB by the human cytomegalovirus gene product UL144. J. Virol. 83, 3581-3590. Alejo, A., Ruiz-Argello, M. B., Viejo, A., Saraiva, M., Fernndez de Marco, M. M., Salguero, J. and Alcami, A. (2009) Adaptation of a transient dominant selection method to the generation of ectromelia virus recombinants: in vivo analysis of ectromelia virus CD30 deletion mutants. PLoS ONE 4(4):e5175. Alcami, A. and Viejo-Borbolla, A (2009) Identification and characterization of virus-encoded chemokine binding proteins. Methods Enzymol. 460, 173-191. Shang, L., Thirunarayanan, N., Viejo-Borbolla, A., Martin, A. P., Bogunovic, M., Marchesi, F., Unkeless, J. C., Ho, Y., Furtado, G. C., Alcami, A., Merad, M., Mayer, L. and Lira, S. A. (2009) Expression of the chemokine binding protein M3 promotes marked changes in the accumulation of specific leukocytes subsets within the intestine. Gastroenterology 137, 1006-1018. Lpez-Bueno, A., Tamames, J., Velzquez, D., Moya, A., Quesada, A. and Alcami, A. (2009) High diversity of the viral community from an Antarctic lake. Science 326, 858-861. Alejo, A. and Alcami, A. (2010) Chemokine binding proteins as therapeutics. In: Leurs, R., Smit, M. and Lira, S. (eds) Chemokine Receptors as Drug Targets. Wiley-VCH, Weinheim, Germany, pp.359-374. Rubio, N., Palomo, M. and Alcami, A. (2010) Interferon a/b genes are upregulated in murine brain astrocytes after Theilers murine encephalomyelitis virus infection. J. Interf. Cyt. Res. 30, 253-262. Viejo-Borbolla, A., Muoz, A., Tabares, E. and Alcami, A. (2010) Glycoprotein G from pseudorabies virus binds to chemokines with high affinity and inhibits their function. J. Gen. Virol. 91, 23-31. Fernandez de Marco, M. M., Alejo, A., Hudson, P., Damon, I. K. and Alcami, A. (2010) The highly virulent variola and monkeypox viruses express secreted inhibitors of type I interferon. FASEB J. 24, 1479-1488. Devlin, J. M., Viejo-Borbolla, A., Browning, G. F., Noormohammadi, A. H., Gilkerson, J. R., Alcami, A. and Hartley, C. A. (2010) Evaluation of immunological responses to a glycoprotein G deficient candidate vaccine strain of infectious laryngotracheitis virus. Vaccine 28, 1325-1332. Alcami, A. and Lira, S. A. (2010) Modulation of chemokine activity by viruses. Curr. Opin. Immunol. 22, 482-487. Alcami A. (2010) The interaction of viruses with host immune defenses. Curr. Opin. Microbiol. 13, 501-502. Viejo-Borbolla, A., Martin, A. P., Muniz, L. R., Shang, L., Marchesi, F., Thirunarayanan, N., Harpaz, N., Garcia, R. A., Apostolaki, M., Furtado, G. C., Mayer, L., Kollias, G., Alcami, A. and Lira, S. A. (2010) Attenuation of TNF-driven murine ileitis by intestinal expression of the viral immunomodulator CrmD. Mucosal Immunol. 3, 633-644. CBMSO 2009-2010

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Home Member of the Editorial Board of Virology Exit Member of the Editorial Board of Journal of Virology Advisor to the World Health Organization Advisory Committee on Variola Virus Research Editor of a special issue of Curr. Opi. Microbiol. on Host-microbe interacions: viruses, vol. 13, August 2010.
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Molecular bases of viral pathogenesis and anti-cancer potential

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The research of the laboratory is mainly focused in the molecular mechanisms operating in viral diseases and in the design of viruses as potential anti-cancer biological tools. We use as experimental models the parvovirus Minute Virus of Mice (MVM) and the alphavirus Sindbis virus (SV). Among those topics recently addressed, or under current research interest, the following two are within the main ones: (i) The regulation of MVM capsid assembly in human cancer cells. We have found that VP2, the major MVM capsid protein, is specifically phosphorylated by Raf-1 (Figure 1), a kinase playing key roles in the development of several common human cancers. The phosphorylation by a mutationactivated Raf-1 facilitates the nuclear transport of VP2 trimers (Figure 1B), a process essential for the viral maturation. This MVM assembly regulation (Figure 1C) may explain most aspects of the parvovirus natural tropism toward human cancer cells, and it does identify a relevant target for possible oncolytic virotherapies. (ii) Translation control of viral mRNA. Our working hypothesis is that translation of viral mRNA may be directing key aspects of virus cycle such as host range and evolution, tropism for cells and tissues, and pathogenesis. Our main goal is to understand how the PKR cellular kinase modulates replication of SV (Figure 2) and MVM through the phosphorylation of translation initiation factor 2 (eIF2). By means of systems biology, we are also characterizing the molecular mechanism used by SV to overcome the antiviral effect of PKR, which involves specific structures in viral mRNA and the use of alternative initiation factors supporting viral translation. The group of Begoa Aguado (*) is focused on Alternative Splicing as a factor contributing to complexity and diversity among primates and other mammals, through the generation of different transcripts, including chimeric and non-coding ones. We are also investigating alternative splicing alterations which can cause disease, such as Rheumatoid Arthritis and Myotonic Dystrophy. We are using new technologies such as nano Q-PCRq, microarrays and Massive Sequencing.
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Figure 1. The Raf1 kinase regulates MVM assembly. (A) Two-dimensional phosphopeptide map of MVM capsid subunits in vitro phosphorylated by Raf-1. (B) Trimers of the MVM capsid protein isolated from human and insect cells differ in their nuclear transport competence. (C) The role of Raf-1 in the nuclear translocation of MVM assembly intermediates.
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Figure 2. The shut-off phenomenon in mouse brain tissues infected with Sindbis virus. Translation of cellular, but not of viral mRNAs, was inhibited due to eIF2 phosphorylation.
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Group Leader: Jos M. Almendral del Ro Scientific Staff: Begoa Aguado Orea (*) Antonio Talavera Dez Ivn Ventoso Bande
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Postdoctoral Fellows: Elena Domingo Gil Esther Grueso Hierro Francisco Hernndez-Torres (*) Graduate Student: Noelia Blanco Menndez Maria Elizalde Arbilla Ren Toribio Lpez Olatz Villate Bejarano (*) Alberto Rastrojo Lastras (*) Raquel Lpez-Dez (*) Undergraduate Students: Marian Fernandez Estevez Julia Wienke (*)
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Sanz MA, Castell A, Ventoso I, Berlanga JJ, Carrasco L. (2009). Dual mechanism for the translation of subgenomic mRNA from Sindbis virus in infected and uninfected cells. PLoS One. 4(3):4772.
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Blanco, AM, L Rausell, B Aguado, M Perez-Alonso, R Artero. (2009). A FRET-based assay for characterization of alternative splicing events using peptide nucleic acid fluorescence in situ hybridization. Nucleic Acids Research 37 e116. Riolobos, L., Valle, N., Hernando, E., Maroto, B., Kann, M., and J. M. Almendral. (2010). Viral oncolysis that targets Raf-1 signaling control of nuclear transport. J. Virol., 84, 2090-2099.
Ventoso, I., Berlanga, J.J., and J. M. Almendral. (2010). Translational control by the Protein Kinase R restricts Minute Virus of Mice infection: role in parvovirus oncolysis. J. Virol., 84, 5043-5051. Toribio R, Ventoso I. Inhibition of host translation by virus infection in vivo. (2010). Proc Natl Acad Sci USA. 107(21):9837-42.
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Home Olatz Villate. Splicing en el MHC de clase III: caracterizacin y expresin de las isoformas del gen NFkBIL1. Estudio de su relacin con artritis reumatoide. Directora, Begoa Aguado. Ren Toribio Lpez. Cambios traduccionales que regula la interaccin virus hospedador. Implicacin en el desarrollo de virus oncolticos. Director, Ivn Ventoso
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Molecular Ecology of Extreme Environments

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Two main lines of research are currently in progress in our group: Molecular Ecology of Extreme Environments: This area of research has the following objectives: - Acidophiles: conventional microbial ecology (enrichment cultures, isolation, physiology), molecular ecology (DGGE, FISH, CARD-FISH, cloning), molecular biology (genomics, metagenomics, proteomics, differential gene expression using DNA arrays) and biotechnology (control of bioleaching, specific metal sequestering and phytoremediation) of extreme acidic environments (Ro Tinto basin, different acidic lakes of the Iberian Pyritic Belt, ro Agrio (Argentina), volcanic areas of Island, Antartica), - Geomicrobiological characterization of extreme environments as habitability models of astrobiological interest: Tinto basin (Mars geochemical analogue), sulfide deposits from Antartica (Mars analogue), Tirez hypersaline lagoon (Europa analogue, in collaboration with I. Marn, associate professor of the Department of Molecular Biology), Uyuni salt lake (Europa analogue, in collaboration with I. Marn), volcanic areas of Island, permafrost areas of Alaska (Mars analogue). - Geomicrobiology of the Iberian Pyritic Belt (IPB) subsurface: different techniques are being implemented in the characterization of the subsurface bioreactor responsible of the extreme acidic conditions of Ro Tinto. This work is done in collaboration with the Centro de Astrobiologa (Origin Project IPBSL) - The line of microbial ecology of anaerobic environments directed by professor J.L. Sanz (UAM) is being developed in the new facilities that the Department of Molecular Biology has in the Biology Building. This collaborative work is centred in the anaerobic activities detected in the different model systems studied by our group (Tinto basin, IPB, Tirez lagoon). Micology, This area of research directed by Dr. Aldo Gonzlez has the following objectives: - Molecular genetics and microbiology of Basidiomicetes (Pleurotus ostreatus as model system). - Use as filamentous fungi as a source of secondary metabolites, lignolytic enzymes and specific sequestering of toxic metals. - Control and elimination of fungi from air-indoor.
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Figura 1. Specific Cr(III) sequestering acidophilic fungi.
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Figura 2. Acidophilic photosynthetic biofilm.

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Group Leader: Ricardo Amils Pibernat Scientific Staff: Aldo Gonzlez Becerra
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Posdoctorales: Moustafa Malki Monika Oggerin de Orube Lourdes Rufo Nieto Javier Ruiz Prez
Graduate Students: Patxi San Martn Uriz Enrique Marn Palma Carlotta Vizioli Irene Snchez Andrea Technical Assistance: Nuria Rodrguez Gonzlez Catalina del Moral Juarez Visiting Scientists: Linda Amaral (MBL, Woods Hole, USA) Jim Field (Arizona State University, USA) Alberto Gonzlez Fiaren (NASA-Ames, USA) Eric Zettler (MBL, Woods Hole, USA)
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Publications
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Fairen, A.G., Dvila, A.F., Duport, L.G., Amils, R., McKay, C.P. (2009) Nature, 459: 398-400. Fairen, A.G., Schulze-Makuch, D., Rodrguez, A.P., Fink, W., Dvila, A., Uceda, E.R., Furfaro, R., Amils, R., McKay, C.P. (2009) Planet. Space Sci., 57: 276-287.
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Daz-Garretas, B., Asensi, A., Rufo, L., Rodrguez, N., Snchez-Mata, D., Amils, R., de la Fuente, V. (2009) Northeastearn Naturalist, 16 : 56-64. Carnicero, D., Daz, E., Escolano, O., Rubinos, D., Ballesteros, O., Barral, M.T., Amils, R., Garca Frutos, F.J. (2009) Adv. Materials Research, 71-73: 677-680. Eugenio, M.E., Carbajo, J.M., Martn, J.A., Gonzlez, and A.E. Villar, J.C. (2009) J. Basic Microbiol. 49(5): 433-440.
Gonzlez-Toril, E., Aguilera, A., Souza-Egipsy, V., Diez, M., Lpez-Pamo, E., Snchez-Espaa, J., Amils, R. (2009) Adv. Materials Research, 71-73: 113-116. Amils, R., Gonzlez-Toril, E., Aguilera, A., Rodrguez, N., Fernndez-Remolar, D., Daz, E., GarcaMoyano, A., Sanz, J.L. (2009) Adv. Materials Research, 71-73: 13-19. Garca-Moyano, A., Gonzlez-Toril, E., Amils, R. (2009) Adv. Materials Research, 71-73: 109-112. Gonzlez-Toril, E; Amils, R; Delmas, RJ, et al. 2009. Biogeosciences, 6(1): 33-44. de la Fuente, V., Rufo, L., Rodrguez, N., Amils, R., Zuluaga, J. (2009) Biol Trace Elem Res, DOI 10.1007/s12011-009-8471-1. Aguilera, A., Souza-Egipsy, V., Gonzlez-Toril, E., Rendueles, O., Amils, R. (2010). Internat. Microbiol., 13: 29-40. DOI: 10.2436/20,1501.01.109. Gonzlez-Toril, E., Aguilera, A., Rodrguez, N., Fernndez-Remolar, D., Gmez, F., Daz, E., GarcaMoyano, A., Sanz, J.L., Amils, R. (2010) Hydrometallurgy, 104: 329-333. Cid, C., Garcia-Descalzo, L., Casado-Lafuente, V., Amils, R., Aguilera, A. (2010) Proteomics, 10: 20262036 DOI 10.1002/pmic.200900592. Souza-Egipsy, V., Aguilera, A., Mateo-Mart, E, Martn-Gago, J.A., Amils, R. (2010). Geomicrobiol. J., 27: 692-706.
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Lalueza, J., Rius, A., Puig, R., Mart, E., Mart, J.F., Rodrguez, N., Amils, R. (2010). Journal of American Leather Chemists Association, 105: 210-221. Amaral-Zettler, L., Zettler, E.R., Theroux, S.M., Palacios, C., Aguilera, A., Amils, R. (20010) ISME J. doi:10.1038/ismej.2010.101.
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Arana-Cuenca, A., Tllez Jurado A., Yage, S., Fermian, E., Carbajo, J.M., Gonzlez, T., Domnguez, A., Villar, J.C., and Gonzlez, A.(2010) Forest Systems 19 (2): 234-240. Eugenio, M.E., Santos, S.M., Carbajo, J.M., Martn, J.A., Martn-Sampedro, R., Gonzlez, A.E., and Villar, J.C. (2010) Bioresources Technology 101(6): 1866-1870. Fairn, A.G., Chevier, V., Abramov, O., Marzo, G.A., Gavin, P., Davila, A.F., Tornabene, L.L. , Bishop, J.L., Roush, T.L., Gross, C., Kneissl, T., Uceda, E.R., Dohm, J.M., Schulze-Makuch, D., Rodrguez, J.A.P., Amils, R., McKay, C.P. (2010) PNAS (USA), doi/10.1073/pnas.1002889107.
Fairen, A. G., Dvila, A.F., Lim, D., Bramall, N., Bonaccorsi, R., Zavaleta, J., Uceda, E.R., Stoker, C.,, Wierzchos, J., Amils, R., Dohm, J.M., Andersen, D., McKay, C. (2010) Astrobiology, 821- 843. DOI:10.1089/ ast. 2009.0440. Fernndez-Remolar, D., Snchez-Romn, M., Amils, R. (2010) Sustainability, 2: 2541-2554, doi:10.3390/su2082541. Gmez; F., Mateo-Mart, E., Prieto.Ballesteros, O., Martn-Gago, J., Amils, R. (2010) Icarus, doi:10.1016/j. icarus.2010.05.027. Carbajosa, S., Malki, M., Caillard, R., Lpez, M.F., Palomares, F.J., Martn-Gago, J.A., Rodrguez, N., Amils, R., Fernndez, V.M., De Lacey, A.L. (2010) Biosensors and Bioelectronics, 26: 877-880. Fernndez-Remolar, D., Prieto-Ballesteros, O., Gmez-Ortiz, D., Fernndez-Sampedro, M., Sarrazin, P., Gailhanou, M., Amils, R. (2010) Icarus, 211: 114-138. Menor-Salvn, C., Tornos, F., Fernndez-Remolar, D., Amils, R. (2010) Earth Planet. Sci. Lett., doi: 10.1016/j.epsl.2010.09.020. Captulos de libros: Cinco captulos de libro. Libros: Amils, R., Segura, J. (2010) Ro Tinto viaje a Marte, ediciones Alfar (Sevilla).
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Patents
Home Amils, R. Malki, M., Fernndez, V., de Lacey, A.L. Ttulo: Electrodo bacteriano aerbico para nodo de una pila de combustible sin mediadores redox ni membrana intercambiadora de protones. N de solicitud: 200701534. Fecha de concesin: 23/12/2009
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Doctoral Theses
Home Eric Zettler, enero 2009, The relationship between environment and the microbial community in a patchwork of geochemical islands, Ricardo Amils, Universidad Autnoma de Madrid.
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Bacterial Cell Division and Antibiotics Resistance

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The objectives of our group are the analysis of the bacterial growth and cell division under diverse molecular approaches, and to study the mechanisms of resistance to the -lactam antibiotics, that have been developed by pathogens of clinical origin. Under this general scheme, we have developed two main aspects. One is the molecular characterization of the CTX-M -lactamases family, their mobilization mechanisms and the ancestral origin of the CTX-M-1 subfamily. Also the identification of PBPs and their role on the resistance mechanisms of anaerobic strains and Gram-negative enteric bacteria was analyzed. A new family of PBPs, belonging to the COG1680, with high homology with class C -lactamases and been involved in morphogenesis, are going to be analyzed in a new project. Based on analysis of peptidoglycan structure of Aeromonas PBP4 mutants, we have proposed a model for induction of the expression of -lactamase mediated by a two component regulatory system. (See joint figure) During cell division, assembly of proteins at a division ring has the effect of constricting the membrane and producing a cell wall septum. The synthesis of a rigid peptidoglycan septum involves a set of dedicated enzymes, as penicillin-binding proteins. We have achieved the purification of several of these enzymes (PBP1B, PBP3, and inactive variants) together with a good number of substrates (including sacculi, as the largest available structure, fragmented peptidoglycan and smaller-sized precursors as lipid II and UDP-muramyl pentapeptide) to assist in the set up of in vitro screening assays.
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Figure 1. Model for beta-lactamase induction in Aeromonas spp. and the role of the blr regulon. Inhibition of PBPs by b-lactams causes an increase in the concentration of the BlrB activatory ligand. This ligand then interacts with BlrB (dotted arrow), causing it to auto-phosphorylate (P in the diagram). This phosphate is transferred to BlrA, which binds to the cre/ blr tag sequence found upstream of all blr regulon gene promoters. The effect of this is to recruit RNA polymerase and activate blr regulon transcription. Known blr regulon genes encode three blactamases, Amp, Cep and Imi, which directly hydrolyse the b-lactam, helping to prevent further peptidoglycan damage. The blr regulon is also known to include non-b-lactamase-encoding genes, e.g. blrD, and it is proposed that their products have a role in protecting the cell from b-lactam challenge through an ancillary mechanism.
Figure 2. DD-endopeptidase activity of AmpH identified by HPLC. Peaks correspond to M4.- NAcGlc-NAcMurtetrapeptide , M5.- NAcGlc-NAcMur-pentapeptide, D45.Disacharide-tetra-pentapeptide, and C.- cefmetazol.
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Group Leader: Juan Alfonso Ayala Serrano Postdoctoral Fellow: Silvia Marina Gonzlez Leiza Graduate Students: Cristian Gustavo Aguilera Rossi Alaa Ropy Mahmoud Sayed Undergraduate Students: Godofrey Cherry Yasemin Ezgi Ertrk Andrs Caballero Laura Vuolo Jose Roberto Angeles Vzquez Nicolas Cordeiro Ana Fernndez Gonzlez Sorelis Urdaneta Fernndez Lucia Lozano Paula Andrea Espinal Mara Margarita Bernal
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Visiting Scientists: Ayelen Patricia Porto (Universidad de Buenos Aires, Buenos Aires, Argentina) Ana Catarina Souza Lopes (Universidade Federal de Pernambuco, Brasil) Bartolome Moya (Universidad Islas Baleares, Mallorca) Judith J. Velasco (Universidad de los Andes, Mrida, Venezuela) -Jzsef Ski University of Szeged, -Elizabeth Nagy University of Szeged, -Gabriella Terhes (University of Szeged, Szeged, Hungary) Sergei Borchsenius (Institute of Cytology, RAS, Saint-Petersburg, Russia)
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D. Korsak, Z. Markiewicz, G. O. Gutkind, and J. A. Ayala. (2010) Identification of the full set of Listeria monocytogenes penicillin-binding proteins and characterization of PBPD2 (Lmo2812). BMC Microbiology, 10:239-251 Ins Bado, Nicols F. Cordeiro, Luciana Robino, Virginia Garca-Fulgueiras, Vernica Seija, Cristina Bazet, Gabriel Gutkind, Juan A. Ayala, and Rafael Vignoli. (2010). Detection of class 1 and 2 integrons, extendedspectrum -lactamases and qnr alleles in enterobacterial isolates from the digestive tract of Intensive Care Unit inpatients. Intern. J. Antimicrob. Agents, 36(5):453-458 A.C.S. Lopes, D. Leal Veras, A.M.S. Lima, R.C. Andrade Melo, and J.A. Ayala. (2010). bla(CTX-M-2) and bla(CTX-M-28) extended-spectrum beta-lactamase genes and class 1 integrons in clinical isolates of Klebsiella pneumoniae from Brazil.. MIOC. 105(2):163-167. Amy E. Tayler, Juan A. Ayala, Pannika Niumsup, Katrin Westphal, Timothy R. Walsh, Bernd Wiedemann, Peter M. Bennett, and Matthew B. Avison. (2010). Induction of beta-lactamase production in Aeromonas hydrophila is responsive to beta-lactam-mediated changes in peptidoglycan composition. Microbiology. 156(Pt 8):2327-35. Porto, Ayeln; Ayala, Juan; Gutkind, Gabriel; and Di Conza, Jos. (2010). A novel OXA-10-like -lactamase is present in different Enterobacteriaceae. Diagnostic Microbiology and Infectious Disease. 66:228-229. M. Macedo-Vias, N. F. Cordeiro, I. Bado, S. Herrera-Leon, M. Vola, L. Robino, R. Gonzalez-Sanz, S. Mateos, F. Schelotto, G. Algorta, J. A. Ayala, A. Echeita and R. Vignoli. (2009). Surveillance of antibiotic resistance evolution and detection of class 1 and 2 integrons in human isolates of multiple resistant Salmonella Typhimurium obtained in Uruguay from 1976 to 2000. International Journal of Infectious Diseases, 13:342-348.
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Profesor Ad Honorem, Facultad de Medicina, Universidad de la Republica, Uruguay Julio, 2009.
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Biotechnology and Genetics of Extreme thermophilic Bacteria

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We use the extreme thermophilic bacteria Thermus thermophilus (Tth) as our main laboratory model. Tth grows quite fast at 70 C, forms colonies on plates, and shows natural competence. As other thermophiles, its enzymes show a greater ability to crystallize than their mesophilic homologues, thus making it an excellent model for Structural Biology. Its ancient phylogenetic origin also adds biological and evolutionary interest to this model. We pursue both biotechnological and basic-science objectives with this model. In biotechnology: i) We overproduce and use thermostable enzymes (thermozymes) for biotransformations in collaboration with external groups specialized in biocatalysis; ii) We evolve enzymes and proteins towards either increasing their thermostability through in vivo selection, or their specific activities at low temperatures; and iii) We develop new genetic tools for its use in thermophiles. In the last two years we have overproduced more than 50 thermozymes (dehydrogenases, estherases, and glycosidases) and carried out selection procedures for the isolation of thermostable mutants of three proteins. In basic-science we study the energy metabolism of Tth, specifically the respiration of nitrogen oxides denitrification-, its genetic control, and its lateral gene transfer (LGT). Through massive sequencing we have shown that the process is catalyzed by three reductases encoded within a denitrification island (DI) that is the subject of frequent LGT events. This DI concentrates information on new type of enzymes that replace the aerobic respiratory complexes I and III, by more simple ones or by bifunctional enzymes acting as reductases-electrons transporter. The DI also encodes new sensory-signal transduction system that allows the organism to select the components of its respiratory chains according to environmental signals. For the next years we will continue these studies and we start new projects on unconventional mechanisms that prevent the LGT between bacterial species.
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Figure 2. Evolving an esterase. (R)-1-phenyl2-propyl acetate docked in the active site of a quadruple mutant (mutant Q) of the Pseudomonas fluorescens esterase I.
Figure 1. The denitrification apparatus of Thermus thermophilus. An scheme of the localization and role of the NADH dehydrogenase (Nrc), and the nitrate (Nar), nitrite (Nir), and nitric oxide (Nor) reductases is shown. Note the role of Nar as electron transporter replacing the complex III. Electrons pathway is shown as red arrows.
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Home Group Leader: Jos Berenguer Carlos Exit Scientific Staff: Aurelio Hidalgo Huertas Postdoctoral Fellows: Daniel Vega Leticia Torres Graduate Students: Federico Acosta Eloy Ferreras Puente Zahra Chahlafi Marcos Almendros Gimenez Laura lvarez Muoz Carlos Bricio Garber No Rigoberto Rivera Yamal Al-Ramahi Gonzlez Martin Hesseler Undergraduate Students: Alba M Sanchez Nio, Akbar Espaillat Fernndez ngel Cantero Camacho Daan Swarts Technical Assistance: Esther Snchez Freire Maria Jos de Soto Lpez Visiting Scientists: Estariette van Heerden Koos Albertin Derek Litthauer Godfrey Tlou Philip Armand Bester Mariana Erasmus Novalanda Betty Mabizela Susana Alarico Hernn Costa
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Larsen,M., Zielinska, D.F., Martinelle, M., Hidalgo, A., Jensen, L., Bornscheuer, U.T. and Hult, K. (2010) Suppression of water as a nucleophile in Candida antarctica lipase B catalysis. ChemBioChem 11,796-801. Cava, F, Hidalgo, A., and Berenguer, J. (2009) Thermus thermophilus as biological model. Extremophiles, 13, 213-231. Schliemann, A., Hidalgo, A., Berenguer, J. and Bornscheuer, U.T (2009). Increased Enantioselectivity by Engineering Bottleneck Mutants in an Esterase from Pseudomonas fluorescens. ChemBioChem 10,29202923 (cover feature). Rocha-Martin, J., Vega D., Cabrera Z, Bolivar JM. Fernandez-Lafuente R., Berenguer J. and Guisan, J.M. (2009) Purification, immobilization and stabilization of a highly enantioselective alcohol dehydrogenase from Thermus thermophilus HB27 cloned in E. coli. Proccess in Biochemistry, 44,1004-1012. Bolivar J.M., Rocha-Martin J., Mateo C., Cava F., Berenguer, J, Vega D. , Fernandez-Lafuente R., Guisan J.M. (2009) Purification and stabilization of a glutamate dehygrogenase from Thermus thermophilus via oriented multisubunit plus multipoint covalent immobilization. Journal of Molecular Catalysis B: Enzymatic 58, 158-163. Almendros M., Sinisterra JV, and Berenguer J. (2009) Thermus thermophilus Strains Active in Purine Nucleoside Synthesis. Molecules 2009, 14, 1279-1287. Bolivar J.M, Rocha-Martin J. Mateo C., Cava F., Berenguer J., Fernandez-Lafuente R., Guisan J. M. (2009) Coating of soluble and immobilized enzymes with ionic polymers: full stabilization of the quaternary structure of multimeric enzymes. Biomacromolecules 10, 742-747 Bolivar J., Mateo, C., Rocha-Martin, Cava F., Berenguer J., Fernandez-Lafuente R., Guisan J. M. 2009. The adsortion of multimeric enzymes on very lowly activated supports involves more enzyme subunits: stabilization of glutamate dehydrogenase from Thermus thermophilus by immobilization on heterofunctional supports. Enzyme Microb. Technol. 44, 139-144. Cava, F., Chahlafi, Z., Alavare, L., and Berenguer, J. (2009) Respiracin y desnitrificacin en Thermus thermophilus. In: Bonete, M. J., and Martnez-Espinosa R. M. (ed) Avances en el metabolismo del nitrgeno. Editorial Club Universitario, Alicante. pp. 173-186.
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Procedimiento de inmovilizacin de una glutamato deshidrogenasa. PCT/ES2008/070166. Nuevo marcador termoestable para la seleccin gentica de Thermus spp P200603279. (Concesin 28-05-2010).
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B6
ASFV: virus models of evasion and protection

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During the last years we have been searching for established cell lines sensitive to the African Swine Fever Virus (ASFV), for the production and titration of virus isolates both from the field or laboratory, as well as for the generation of virus recombinants with specific genes deleted. These deletion mutants facilitate the study of the role of several ASFV genes involved in the evasion of the antiviral response of the cell during virus infection, and may serve as attenuated virus models in the generation of possible vaccines to induce protective immunity against ASF. So, we have confirmed the role of the ASFV lectin (EP153R gene) in the modulation of the expression of MHC-I antigens in the membrane of the infected cell, and we are also inactivating several viral genes in a partially-attenuated ASFV isolate (NHV), able to induce protection against the virulent L60 isolate, but still retaining a residual virulence unacceptably high to be used as a vaccine. The porcine cell lines sensitive to ASFV infection are also being used for the determination of the level of induction of selected cytokines by ASFV isolates with various degrees of virulence, searching for a possible virulence/attenuation profile, which might result in a remarkable reduction of the in vivo infections required to determine the degree of virulence of the virus isolates. Another objective of our Group is the optimization of the use of non-conventional antivirals as the lauryl gallate (LG) in the prevention and/or treatment of viral diseases of clinical and veterinarian importance. We have demonstrated its low cytotoxicity and its efficiency in the inhibition of the virus production in several models (ASFV, influenza, herpes,...) in cells infected in the laboratory, and we pretend to extend the study of the protective level provided by the LG in in vivo infections.
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Figure 1. Interaction EP153R - SLA-I. Residues involved

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Home Group Leader: ngel L. Lpez Carrascosa Exit Postdoctoral: Patricia de Len Valds Undergraduated Student: Alba Martnez Flrez Technical Assistance: Maria Jos Bustos Snchez
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Hurtado, C., Bustos, M. J. and Carrascosa, A. L. (2010). The use of COS-1 cells for studies of field and laboratory African swine fever virus samples. J. Virol. Methods 164, 131-134.
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Hurtado, C., Bustos, M. J., Granja, A. G., de Len, P., Sabina, P., Lopez-Vias, E., Gomez-Puertas, P., Revilla, Y., and Carrascosa, A. L. (2010). The African swine fever virus lectin EP153R modulates the surface membrane expression of MHC class I antigens. Arch. Virol. DOI: 10.1007/s00705-010-0846-2.
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B7
Bacterial morphogenesis
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Home Our main interest is the investigation, at the molecular and structural levels, of the bacterial cell wall (sacculus or peptidoglycan layer) as the primary morphogenetic element of the prokaryotic cell. We are presently working on cell development, adaptation and differentiation of appendages (prostheca, flagella, pili and hold-fast) in alfa-proteobacteria, mainly in Asticcacaulis biprosthecium, and other bacterial groups of complex morphology. We want to extend our experience in simple cell-cycle organisms as Escherichia coli, to more complex bacteria as A. biprosthecium. This species presents a dimorphic cell cycle with a mobile flagellated form (swarmer cell) which eventually differentiates into a sessile form displaying two laterally opposed prostheca. Only the sessile form is division proficient, and generates a sessile and a swarmer cell upon division. At present we are investigating how the metabolism and structure of the cell wall are modified in accordance with the cell cycle differentiation processes, and in relation with the generation of cell appendages (prostheca, flagella, and hold-fast) at precise places and times in the cell cycle. Recently we started a new line of work centred on the discovery of a novel regulatory mechanism of cell wall metabolism in Vibrio cholerae and other bacteria. The mechanism is mediated by the production and release of specific D-amino acids, is part of the general stress response mechanism, and has the potential to act as an inter and intra specific signalling system. We are also involved in cooperative projects concerning structural and biochemical aspects of cell wall metabolism in other organisms, Listeria monocitogenes or Leptospira biflexa, associated to the development of symbiotic or pathologic relations and to peculiar morphologies.
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Figure 1. Asticcacaulis biprosthecium cells visualized by confocal microscopy.
Figure 2. Actively growing (A) and resting (B) cells of Vibrio cholera as visualized by ESM.
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Group Leader: Miguel Angel de Pedro Montalbn Graduate Students: Said Taimani Undergraduate Students: Marisela Domnguez Domnguez Mara Hidalgo Garca Irene Cartas
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Lam, H, Oh, D.C., Cava, F., Takacs, C.N., Clardy, J., de Pedro, M.A. and Waldor, (2009) D-amino acids govern stationary phase cell wall remodeling in bacteria. Science 325:1552-1555.
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de Pedro, M.A. (2009). Peptidoglycan (Murein), In Encyclopedia of Microbiology. (Moselio Schaechter, Ed.), pp. 453-469, Oxford: Elsevier.
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B8
Genetic variability of RNA viruses

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The main interest of our group is to understand the molecular bases of RNA virus extinction by lethal mutagenesis, and to explore antiviral protocols using mutagenic agents and antiviral inhibitors. We have documented a double mutagenic and inhibitory activity of 5-fluorouracil on foot-and-mouth disease virus (FMDV), and have characterized FMDV mutants resistant to the mutagenic purine analogue ribavirin. Specifically, we have described an amino acid substitution in the FMDV polymerase which avoids the deleterious activity of ribavirin by means of a modulation of the mutation types produced by the drug. Virus survival through modulation of mutation types, without an alteration of the general copying fidelity of the polymerase, constitutes a new mechanism of resistance to a mutagenic agent. Despite the presence of mutagen-resistance mutations, virus extinction can be achieved with alternative mutagenic treatments. Contrary to what has been established for classic antiviral treatments, a sequential administration of an inhibitor first, and then of a mutagenic agent can be more effective for virus extinction than the administration of the two drugs simultaneously. With regard to the understanding of the biological implications of quasispecies behavior, we have characterized a competition-colonization dynamics among FMDV subpopulation that arose in cell culture through diversification of a biological clone of the virus. Our laboratory has participated in collaborations with various teams on theoretical and experimental virology, on structural studies with the FMDV polymerase, and biological implications of quasispecies, including new vaccine designs. Such collaborations can be identified by the names of the authors of the publications included in the present summary.
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Figure 1. Antiviral strategy based on the extinction of viruses by increasing their mutation rates during replication.

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Group Leader: Esteban Domingo Solans Postdoctoral Fellows: Celia Perales Viejo Vernica Martn Garca Armando Arias Esteban Julie Sheldon
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Graduate Students: Rubn Agudo Torres Marta Sanz-Ramos Rojo Samuel Ojosnegros Martos Hctor Moreno Borrego Hctor Tejero Franco Ignacio de la Higuera Ana M Ortega Prieto Technical Assistance: Ana Isabel de vila Lucas Eva Garca Cueto Isabel Gallego Jimnez M Eugenia Soria Benito
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Publications
Agudo, R. Arias, A. and Domingo, E. (2009). 5-Fluorouracil in lethal mutagenesis of foot-and-mouth disease virus. Future Medicinal Chemistry, 1(3), 529-539. Domingo, E. and Wain-Hobson, S. (2009). The 30th anniversary of quasispecies. Meeting on quasispecies: past, present and future. EMBO Reports, 10, 444-448. Emonet, S.F., de la Torre, J.C., Domingo, E. and Sevilla, N. (2009). Phylogeny/molecular taxonomy and evolution or Arenaviruses. Infection Genetics and Evolution, 9, 417-429. Perales, C., Agudo, R. and Domingo, E. (2009). Counteracting quasispecies adaptability: extinction of a ribavirin resistant virus mutant by an alternative mutagenic treatment. PLoS One, 4(5): e5554.doi: 10.1371/journal.pone.0005554. Escarms, C., Perales, C. and Domingo, E. (2009). Biological effect of Mullers ratchet. Distant capsid site can affect picornavirus protein processing. J. Virol. 83, 6748-6756. Domingo, E. (2009). Nueva gripe humana de origen porcino. Cunto de nuevo? Investigacin y Ciencia. 393, 10-12. Ferrer-Orta, C., Agudo, R., Domingo, E. and Verdaguer, N. (2009). Structural insights into replication and elongation processes by the FMDV RNA-dependent RNA polymerase. Curr. Op. in Structural Biol. 19 (6), 752-8. Perales, C., Agudo, R., Tejero, H., Manrubia, S.C. and Domingo, E. (2009). Potential benefits of sequential inhibitor-mutagen treatments of RNA virus infections. PLoS Pathogens. 5 (11), e1000658. Domingo, E. (2009). Quasispecies. From molecular Darwinism to viral diseases. Contributions to Science. 5(2), 161-168. Ojosnegros, S., Beerenwinkel, N., Antal, T., Nowak, M.A., Escarms, C. and Domingo, E. (2010). Competition-colonization dynamics in an RNA virus. Proc. Natl. Acad. Sci. USA. 107(5), 2108-12. Domingo, E. (2010). The great evolutionary potential of viruses. The 1918 flu as a paradigm of disease emergence. In: M.I. Porras and R. Davies, ed. Emerging Infection, Emergent Meanings: The Spanish Influenza Pandemic of 1918-1919. University of Rochester Press, Rochester, New York. 107, 2108-2112. Domingo, E. (2010). Mechanisms of viral emergence. Veterinary Research, 41(6): 38. Martn, V. Abia, D., Domingo, E. and Grande-Prez, A. (2010). An interfering activity of LCMV associated with enhanced mutagenesis. J. Gen. Virol., 91, 990-1003. Ojosnegros, S., Beerenwinkel, N. and Domingo, E. (2010). Competition-colonization dynamics: an ecology approach to quasispecies dynamics and virulence evolution in RNA viruses. Communicative & Integrative Biology, 3 (4), 333-6. Domingo, E., Perales, C., Agudo, R., Arias, R., Escarms, C., Ferrer-Orta, C. and Verdaguer, N. (2010). Mutation, quasispecies and lethal mutagenesis. In: E. Ehrenfeld, E. Domingo and R.P. Roos, eds. The Picornaviruses. ASM Press, Washington, D.C., pp.197-211. Manrubia, S.C, Domingo, E. and Lzaro, E. (2010). Pathways to extinction: beyond the error threshold. Phil. Trans. R. Soc. B., 365(1548), 1943-52. Ferrer-Orta, C., Sierra, M., Agudo, R., de la Higuera, I., Arias, A., Prez-Luque, R., Escarms, C., Domingo, E. and Verdaguer, N. (2010). Structure of foot-and-mouth disease virus mutant polymerases with reduced sensitivity to ribavirin. J. Virol., 84(12), 6188-99. Bordera, A.V., Lorenzo-Redondo, R., Pernas, M., Casado, C., lvaro, T., Domingo, E. and Lpez-Galndez, C. (2010). Initial fitness recovery of HIV-1 is associated with quasispecies heterogeneity and can occur without modifications in the consensus sequence. PLoS One, 5(4), e10319. Martn-Acebes, M.A., Herrera, M., Armas-Portela, R., Domingo, E. and Sobrino, F. (2010). Cell density-dependent expression of viral antigens during persistence of foot-and-mouth disease virus in cell culture. Virology, 403, 47-55. Rodrguez-Calvo, T., Ojosnegros, S, Sanz-Ramos, M., Garca-Arriaza, J., Escarms, C., Domingo, E. and Sevilla, N. (2010). New vaccine design based on defective genomes that combines features of attenuated and inactivated vaccines. PLoS One, 5(4), e10414. Arias, A., Perales, C., Escarms, C. and Domingo, E. (2010). Deletion mutants of VPg reveal new cytopathology determinants in a picornavirus. PLoS One, 5(5), e10735. Agudo, R., Ferrer-Orta, C., Arias, A., de la Higuera, I., Perales, C., Prez-Luque, R., Verdaguer, N. and Domingo, E. (2010). A multi-step process of viral adaptation to a mutagenic nucleoside analogue by modulation of transition types leads to extinction-escape. PLoS Pathog. 6(8) pii: e1001072. Perales, C., Lorenzo-Redondo, R., Lpez-Galndez, C., Martnez, M. A., and Domingo, E. 2010. Mutant spectra in virus behavior. Future Virology 5(6), 679-698.
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N. Sevilla, E. Domingo, C. Escarms, S. Ojosnegros, J. Garca-Arriaza, M. Sanz-Rojo, T. Rodrguez (2009) Vacuna atenuada para la fiebre aftosa. PCT1641.49. Entidades titulares: CSIC (70%), Instituto Nacional de Investigaciones Agrarias (30%). E. Domingo, R. Agudo, H. Tejero, S.C. Manrubia, C. Perales (2009) Tratamiento antiviral. P200930482. Entidades titulares: CSIC (41%), Instituto Nacional de Tcnica Aeroespacial (18%), Universidad Complutense de Madrid (18%), Centro de Investigaciones Biomdicas en Red de Enfermedades Hepticas y Digestivas (CIBERehd) (23%).

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Doctoral Theses
Home Samuel Ojosnegros Martos (2009) Dinmica evolutiva de virus RNA. Universidad Autnoma de Madrid. Director: Esteban Domingo Solans. Exit Rubn Agudo Torres (2009) Caracterizacin de las protenas del virus de la fiebre aftosa implicadas en respuesta a mutagnesis letal por anlogos de nucletido. Universidad Autnoma de Madrid. Director: Esteban Domingo Solans. Marta Sanz-Ramos Rojo (2009) Dinmica de cuasiespecies del virus de la fiebre aftosa in vivo. Universidad Autnoma de Madrid. Director: Esteban Domingo Solans.
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B9
Gene expression in Streptomyces and yeasts

Research Summary
Home Streptomyces and yeasts produce a high number of molecules with biotechnological value. Our objective is to study the expression molecular mechanism of proteins implicated in some of this biosynthetic process, and to obtain new molecules with possible industrial applicability. During the last two years, we have been studying the biosynthetic cluster of the nucleoside antibiotic A201A (ata) from Streptomyces capreolus, and some yeast proteins with glycosyltransferase activity able to produce prebiotic oligosaccharides. The glycosyl residues constitute about 44% of the A201A total mass, which must be related with the biological-pharmacological properties of the antibiotic. We have characterized some genes involved in the incorporation and modification of these residues using different heterologous expression systems. The biosynthesis of novel molecules with antibiotic activity will be attempted based on the low substrate specificity previously described for the related glycosyltransferases. We are studying several proteins from the Xanthophyllomyces, Schwanniomyces and Rhodotorula yeasts genera, which produce different types of prebiotic oligosaccharides. All the analysed proteins are hydrolases that show transferase activity, included within family 32, 31, 1 and 2 of the glycosylhydrolases. To know the structure-function-specificity relationships of these proteins are one of our objectives. We are carrying out structural studies of these proteins (3-D structure of same of them has been resolved), residue substitutions, and applying directed molecular evolution techniques in order to increase/ modify their transglycosylase activity, and to improve their biotechnological utility. International patents of most of these proteins have been obtained and a method for its immobilization on solid support has been developed. We seek to scale up to industrial level the enzyme production and the generated products.
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Figure 2. Oligomerization pattern of the Ftase from Schwanniomyces occidentalis on a this yeast culture.
Figure 1. (A) Close up view of the active site in Schwanniomyces occidentalis Ffase in a complex with fructose, 1-kestose (green; left) and the 6-kestose (yellow; right). (B) The fructan 1-exohydrolase IIa from Cichorium intybus (CiFEH) complexed with 1-kestose and (C) the invertase from Thermotoga maritima complexed with raffinose. In (A), the active-site of Ffase (blue) is also shaped by the adjacent subunit (orange).
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Group Leader: Antonio Jimnez / Mara Fernndez Lobato Graduate Students: Miguel de Abreu Felipe Miguel lvaro Benito Dolores Linde Lpez Patricia Gutirrez Alonso Brian Molloy Galiana
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Undergraduate Students: David Gonzlez Prez Marta Estvez Canales Hugo Muoz Technical Assistance: Asuncin Martn Redondo Visiting Scientists: Vctor Cifuentes (Chile) Marcelo Baeza (Chile)
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Linde, D., Macias, I., Fernndez-Arrojo, L., Plou, F.J., Jimnez, A. and Fernndez-Lobato, M. (2009) Molecular and biochemical characterization of an fructofuranosidase from Xanthophyllomyces dendrorhous. App Env Microbiol. 75(4), 1065-1073.
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Gutirrez-Alonso, P., Fernndez-Arrojo, L., Plou, F.J. and Fernndez-Lobato, M. (2009) Biochemical characterization of a b-fructofuranosidase from Rhodotorula gracilis with transfructosylating activity. FEMS Yeast Research. 9, 768-773. Polo, A., lvaro-Benito, M., Fernndez-Lobato, M. and Sanz-Aparicio, J. (2009) Crystallization and preliminary X-ray diffraction analysis of thefructofuranosidase from Schwanniomyces occidentalis. Acta Cryst. 65, 1162-1165.
Baeza, M., Retamales, P., Sepulveda D., Lobato P., Jimenez A. and Cifuentes V. (2009) Isolation, characterization and long term preservation of mutant strains of Xanthophyllomyces dendrorhous. J. Basic Microbiol. 49, 135-141. Alvaro-Benito, M., Polo, A., Gonzlez, B., Fernndez-Lobato, M. and Sanz-Aparicio, J. (2010) Structural and kinetic analysis of Schwanniomyces occidentalis invertase reveals a new oligomerization pattern and the role of its supplementary domain in substrate binding. J. Biol. Chem. 285 (18), 13930-14941. Polo, A., Linde, D., Estvez, M., Fernndez-Lobato, M. Julia Sanz-Aparicio, J.. (2010). Crystallization and preliminary X-ray diffraction analysis of the fructofuranosidase from Xanthophyllomyces dendrorhous. Acta Cryst. 66, 1441-1444. lvaro-Benito, M., de Abreu, M., Portillo, F., Snz-Aparicio, J. and Fernndez-Lobato, M. (2010). New insights into the fructosyltransferase activity of Schwanniomyces occidentalis -fructofuranosidase emerges from a non-conventional codon usage and directed mutation. App. Env. Microbiol. 76 (22), 7491-7499.
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Patents
L. Fernndez Arrojo, F. Plou, A. Ballesteros, M. Alcalde, P. Gutierrez Alonso, M. FernndezLobato (2009). Biocatalizador inmovilizado basado en alginato para la biotransformacin de carbohidratos. N P200930001. PCT-ES2010/070104 (24 -2- 2010). Titular: CSIC-UAM. M. Fernndez-Lobato, M. Alvaro Benito (2009). Fructofuranosidasa mejorada genticamente para la obtencin del prebitico 6-kestosa. N P0200930929 (29-12-2009). Titular: UAM.
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Doctoral Theses
Home Brian Molloy Galiana (2010). Expresin heterloga del cluster biosinttico del antibitico A201A y estudio de las posibles glicosiltransferasas implicadas en su biosntesis. Universidad Autnoma de Madrid. Director: Mara Fernndez-Lobato. Mara Dolores Linde Lpez (2010). Caracterizacin bioqumica, molecular y estructural de una b-fructofuranosidasa con capacidad trasferasa de la levadura Phaffia rhodozyma aplicable a la produccin de oligosacridos prebiticos. Universidad Autnoma de Madrid. Director: Mara Fernndez-Lobato.
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B10
Virus Engineering
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We use protein engineering techniques for the study of structure-function relationships in viruses and their capsids, and for the design of modified viral particles for nanobiotechnological applications (Mateu (2011). Prot.Eng.Des.Sel. 24, 53-63). Scientific relevance and technological implications: A better knowledge of poorly known stages of the viral cycle, including virus assemby, conformational dynamics and disassembly; design of new vaccines, antivirals and nanoparticles for targeted drug delivery.
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Research Summary Staff Publications Other Activities Patents Doctoral Theses
Some recent scientific results: i) Using protein engineering, the thermal stability of footand-mouth disease virus (FMDV) against dissociation into subunits has been increased. We are currently investigating the molecular basis of such thermostabilization, and the possibility of using the modified virions as improved vaccines. ii) We have carried out an extensive mutational analysis of compensatory mutations in the FMDV capsid, and are currenly investigating the molecular bases of these effects. iii) In collaboration with P.J. de Pablo y J. Gmez (Dept. Physics of Condensed Matter, UAM) we have used the minute virus of mice (MVM) to investigate the role of capsid pores and cavities in the mechanical properties of the viral particle, using atomic force microscopy (a technique we are currently using in our lab). We have obtained evidence that the remarkable mechanical properties found may have been evolutionarily selected to optimize the resistance of the virus against physical agents, while at the same time allowing the conformational changes needed for infectivity. iv) In collaboration with J.L.Neira (CBMC) y M.A.Martnez (IRSI-Caixa), we are investigating the capacity of different synthetic peptides and other molecules to inhibit human immunodeficiency virus assembly and infectivity. These studies may be important for the design of new anti-HIV agents. v) In collaboration with G.Rivas (CIB), we have used two model viral systems to show that, in physiological macromolecular crowding conditions, the inhibitory activity of small-size antiviral compounds may be reduced.
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Virus Engineering
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Figure 1. Topographic image of an individual MVM particle in liquid, obtained in our laboratory by atomic force microscopy. Immediately after the image is obtained, the microscope is used to apply a force on the particle; this allows the determination of its mechanical elasticity.
Figure 2. Location on the FMDV capsid structure (A) of a collecion of lethal mutations at the interfaces between pentameric subunits (B, colored white), and of the different compensatory mutations that restored infectivity (C, colored green or orange).
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Virus Engineering
Home Group Leader: Mauricio Garca Mateu Exit Postdoctoral: Rebeca Prez Fernndez Graduate Students: Rebeca Bocanegra Rojo Milagros Castellanos Molina Inmaculada Lpez Prez Pablo Jos Prez Carrillo Vernica Rincn Forero Technical Assistance: Miguel ngel Fuertes Villadangos Alicia Rodriguez Huete
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Virus Engineering
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Carrasco, C., Douas, M., Miranda, R., Castellanos, M., Carrascosa, J.L., Mateu, M.G., Marqus, M. and de Pablo, P.J. (2009). Action of capillary forces of water confined at the nanoscale during dessication of viruses. Proc. Natl. Acad. Sci. USA 106, 5475-5480. (A) Serena, P.A., Douas, M., Marqus, M.I., Carrasco, C., de Pablo, P.J., Miranda, R., Carrascosa, J.L., Castellanos, M. and Mateu, M.G. (2009). MC simulations of water meniscus in nanocontainers: explaining the collapse of viral particles due to capillary forces. Phys. Status Solidi C 6, 2128-2132. Mateu, M.G. (2009). Capsid protein interactions in human immunodeficiency virus type 1 assembly. FEBS J. 276, 6098-6109. Luna, E., Rodriguez-Huete, A., Rincn, V., Mateo, R. and Mateu, M.G. (2009). A systematic study of the genetic response of a variable virus to the introduction of deleterious mutations in a functional capsid region. J.Virol. 83, 10140-10151. Martn-Acebes, M., Rincn, V, Mateu, M.G. and Sobrino, F. (2010). A single amino acid substitution in the structural protein VP3 of foot-and-mouth disease virus can increase acid-lability and confer resistance to acid-dependent uncoating inhibition. J.Virol. 84, 2902-2912. Domenech, R., Abin, O., Bocanegra, R., Correa, J., Sousa-Herves, A., Riguera, R., Mateu, M.G., Fernndez-Mega, E., Velzquez-Campoy, A. and Neira, J.L. (2010) Dendrimers bind the homodimerization interface of the capsid protein of HIV-1. Biomacromolecules 11, 2069-2078.
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Virus Engineering
Other Activities
Home Mauricio G. Mateu, member of the Editorial Board of Virus Research Exit

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Virus Engineering
Patents
OEPM Patent n 2 323 929 (Foot-and-mouth disease vaccine). Granted June 2010.
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Virus Engineering
Doctoral Theses
Eva Luna Garca (2010). Aproximaciones a la obtencin de cpsidas ms estables del virus de la fiebre aftosa y estudio de la generacin de mutaciones compensatorias. Universidad Autnoma de Madrid. Director: Mauricio G. Mateu.
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B11
Effects of extrachromosomal elements on behaviour of its host Bacillus

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So-called mobile genetic elements (MGE), e.g. phages, plasmids, transposons and ICEs, can be transferred horizontally between cells and affect the genetic make-up and hence the behaviour of bacteria. Accordingly, horizontal gene transfer (HGT) has a crucial role in microbial evolution and has important implications in a myriad of environmental and public health problems. For instance, HGT is mainly responsible for the emergence and rapid dispersion of antibiotic resistance. Little is known, especially in Gram positive bacteria, about the mechanisms by which MGE exert their behavioural effects on their host or on regulation of their mobility. A better understanding of these issues is warranted to face important threats of for instance antibiotic resistance. In our laboratory we study these issues using as host Bacillus subtilis and we limit the MGE to plasmids and phages. We use B. subtilis because (i) it is probably the best studied Gram-positive bacterium; (ii) it is non-pathogenic; (iii) it is amenable to genetic manipulation due to its ability to develop natural competence; and (iv) B. subtilis is related to pathogenic/fastidious bacteria like Bacillus anthracis, B. cereus and, although more distantly, to Listeria monocytogenes. For some phages it has been described that they alter the behaviour of B. subtilis upon infection. However, neither sequence nor mechanistic information of how these phages affect behaviour of their infected host is available. We are studying these focussing on two phages. About 20% of the natural isolates of B. subtilis contain an endogenous plasmid. Most large plasmids (>10 kb) can be transferred to other cells via the process of conjugation. We are the first to have sequenced two large Bacillus plasmids and have identified genes involved in conjugation as well as others that probably affect behavioural processes of the host. We are now analyzing these to unravel regulation of the conjugation process and to gain insight in the way plasmid-located genes alter the life style of the host.
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Figure 1. Phase contrast microscopy image of sporulating B. subtilis cells harbouring conjugative plasmid pLS20.
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Figure 2. Genetic map of B. pumilus sporulation inhibiting plasmid p576.
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Home Group Leader: Wilfried J.J. Meijer Exit Graduate Students: Praveen K. Singh Gayetri Ramachandran
Undergraduate Students: Esther Serrano Sandra Ballestero Beltrn Technical Assistance: Adriana Marulanda Aguirre
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Singh, P. K., Ballestero-Beltrn, S., Ramachandran, G. and Meijer, W.J.J. (2010) Complete nucleotide sequence and determination of the replication region of the sporulation inhibiting plasmid p576 of Bacillus pumilus NRS576. Res. Microbiol. 161, 772-782.
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Muoz-Espn, D., Daniel, R., Kawai, Y., Carballido-Lpez, R., Castilla-Llorente, V., Errington, J.*, Meijer, W.J.J.*, and Salas, M.* *: contributed equally. (2009) The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria. Proc. Natl. Acad. Sci. USA. July 27 Epub ahead of print. Castilla-Llorente, V., Meijer, W.J.J., and Salas, M. Differential Spo0A-mediated effects on transcription and replication of the related Bacillus subtilis phages Nf and f29 explain their different behaviours in vivo. (2009) Nucleic Acids Res. 37: 4955-4964. Castilla-Llorente, V., Salas, M., and Meijer, W.J.J. (2009) Different responses to Spo0A-mediated suppression of the related Bacillus subtilis phages Nf and f29. Environ. Microbiol. 11: 1137-49.
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Human immunodeficiency virus reverse transcriptase and antiretroviral therapy

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The human immunodeficiency virus (HIV) is a retrovirus that infects cells of the immune system, and is the etiological agent of AIDS. Nowadays, treatment of HIV-infection involves the use of inhibitors of retroviral enzymes such as the reverse transcriptase (RT), protease and integrase; as well as drugs targeting viral entry. Combination therapies including two or three RT inhibitors have been rather successful in the clinic, and play a key role in the management of HIV-infected individuals. However, the emergence of resistant viruses, the observed cross-reactivity between the inhibitors, and unwanted secondary effects are major hurdles towards their long-term efficacy.
We are interested in therapeutic targets for HIV, emphasizing on the role of the viral RT. HIV RT plays a pivotal role in the replication of the viral genomic RNA. Recently, our efforts have been directed towards two major goals: (1) understanding the role of different amino acids in the nucleotide specificity of the enzyme, as well as in its fidelity of DNA synthesis; and (2) the elucidation of molecular mechanisms involved in RT inhibitor resistance and the analysis of the contribution of different amino acid substitutions in the acquisition of drug resistance. Nucleotide specificity studies are relevant in drug resistance but are also important to develop useful tools in molecular biology. Thus, we are trying to obtain more stable and faithful RTs, with biotechnological applications (for example, in the analysis of gene expression). Understanding the role of different residues in RT function (both on DNA polymerase activity, but also in modulating RNase H function and reverse transcription initiation) should help us to design novel strategies for treating HIV infection.
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Figure 1. Crystal structure of HIV-1 reverse transcriptase complexed with an RNA/DNA template-primer, showing (in purple) thumb subdomain residues that modulate nucleoside analogue resistance
Figure 2. ATP-mediated excision kinetics of AZT-monophosphate from blocked primer-template DNA/DNA complexes by wild-type and mutant HIV-1 reverse transcriptases.
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Group Leader: Luis Menndez Arias Postdoctoral Fellows: Mar lvarez Garca Tania Matamoros Grande Graduate Students: Vernica Barrioluengo Fernndez Gilberto J. Betancor Quintana Mnica Kisic Aguirre Undergraduate Students: Raquel N. Afonso Lehmann Daniela Barbieri Yasemin Ezgi Ertrk Luis Goslbez Cisneros-Miret Cristina Jimnez Snchez
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lvarez, M., Matamoros, T. and Menndez-Arias, L. (2009) Increased thermostability and fidelity of DNA synthesis of wild-type and mutant HIV-1 group O reverse transcriptases. J. Mol. Biol. 392, 872-884. De Mendoza, C., Anta, L., Garca, F., Prez-Elas, M.J., Gutirrez, F., Llibre, J.M., Menndez-Arias, L., Dalmau, D., Soriano, V., on behalf of Platform for Drug Resistance of the Spanish AIDS Research Network (2009) HIV-1 genotypic drug resistance interpretation rules 2009 Spanish guidelines. AIDS Rev., 11, 39-51. Garriga, C., Prez-Elas, M.J., Delgado, R., Ruiz, L., Prez-lvarez, L., Pumarola, T., Lpez-Lirola, A., Gonzlez-Garca, J. and Menndez-Arias, L., on behalf of the Spanish Group for the Study of Antiretroviral Drug Resistance (2009) HIV-1 reverse transcriptase thumb subdomain polymorphisms associated with virological failure to nucleoside drug combinations. J. Antimicrob. Chemother. 64, 251-258.
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Matamoros, T., Nevot, M., Martnez, M.A. and Menndez-Arias, L. (2009) Thymidine analogue resistance suppression by V75I of HIV-1 reverse transcriptase: Effects of substituting Valine 75 on stavudine excision and discrimination. J. Biol. Chem. 284, 32792-32802. Menndez-Arias, L. (2009) Mutation rates and intrinsic fidelity of retroviral reverse transcriptases. Viruses 1, 1137-1165; doi:10.3390/v1031137 Puertas, M.C., Buzn, M.J., Artese, A., Alcaro, S., Menndez-Arias, L., Perno, C.F., Clotet, B., CeccheriniSilberstein, F. and Martinez-Picado, J. (2009) Effect of the human immunodeficiency virus type 1 reverse transcriptase polymorphism Leu-214 on replication capacity and drug susceptibility. J. Virol. 83, 7434-7439. Betancor, G., Puertas, M.C., Nevot, M., Garriga, C., Martnez, M.A., Martinez-Picado, J. and MenndezArias, L. (2010) Mechanisms involved in the selection of HIV-1 reverse transcriptase thumb subdomain polymorphisms associated with nucleoside analogue therapy failure. Antimicrob. Agents Chemother. 54, 4799-4811. Clotet, B., Menndez-Arias, L., Schapiro, J. M., Kuritzkes, D., Burger, D., Telenti, A., Brun-Vezinet, F., Geretti, A. M., Boucher, C. A. and Richman, D. D. (eds.) (2010) Guide to management of HIV drug resistance, antiretrovirals pharmacokinetics and viral hepatitis in HIV infected subjects, 10th Edition, Fundaci de Lluita contra la SIDA, Barcelona, Spain, 422 pp. Menndez-Arias, L. (2010) Special issue: Retroviral enzymes. Viruses 2, 1181-1184 (editorial). Menndez-Arias, L. (2010) Molecular basis of human immunodeficiency virus drug resistance: An update. Antiviral Res. 85, 210-231.
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Antirretroviral / human immunodeficiency virus reverse transcriptase and antireOther Activities

Member of the Editorial Boards of Antiviral Therapy, Viruses and Timely Topics in Medicine: AIDS Cyber Journal.
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Editor of a special issue of the on-line journal Viruses dedicated to Retroviral enzymes (2009/2010). Member of the formation and training panel of the Spanish AIDS Research Network, and co-organizer of its Third Training Symposium (San Lorenzo de El Escorial, October 7-9, 2009).
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L. Menndez Arias, T. Matamoros, M. lvarez (2010). Retrotranscriptasa del VIH-1 de grupo O modificada. Ref.: PCT/ES2010/070320.
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Mnica Kisic Aguirre (2010). Mecanismos moleculares de resistencia e hipersensibilidad a frmacos antirretrovirales mediados por deleciones en la horquilla 3-4 de la retrotranscriptasa del virus de la inmunodeficiencia humana tipo 1. Universidad Autnoma de Madrid. Director: Luis Menndez Arias.
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B13
African swine fever virus

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Repair of African swine fever virus (ASFV) DNA. Presently, the objective of this work is to study the biological function of the reparative DNA polymerase, pol X, of ASFV, which participates, together with the viral AP endonuclease and DNA ligase, in a base excision repair (BER) pathway. Our studies show that pol X is required for virus replication in its natural host cell, the swine macrophage, and protects the infected cell against oxidizing genotoxic agents. Moreover, pol X is necessary to maintain genome stability, by repairing mutations introduced in the viral DNA during the infection, which underlines the importance of its in vivo function.
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ASFV morphogenesis. The biogenesis of ASFV membranes is one of the most interesting and controversial subjects in the study of virus morphogenesis. In order to undertake these studies, we are using ASFV recombinants that inducibly express membrane structural proteins localized in the inner envelope of the virus particle.We have recently identified a new morphogenetic intermediate constituted by membrane helicoidal structures that are precursors of icosahedral viral particles. To get a better knowledge of the structure of these helicoidal intermediates, we are presently using electron tomography and other advanced techniques in electron microscopy for the resolution of three-dimensional structures. Another objective of our studies on virus morphogenesis is to understand the regulation of ASFV polyproteins pp220 and pp62 processing. The polyproteins are major components of the viral core, being their proteolytic processing essential for virus maturation. Our results indicate the existence of redox mechanisms that might operate during the infective cycle by regulating the activity of the viral protease that catalyzes the processing of polyproteins.
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Figure 1. Serial section analysis of a viral factory showing a helicoidal membrane structure with an associated icosahedral particle. Right panel, reconstruction of the helicoidal structure. The components of each strand in the different sections are identically colored.
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Home Group Leader: Maria Luisa Salas Falgueras Exit Postdoctoral: Modesto Redrejo Rodrguez Cristina Surez Garca Graduate Students: Cristina Surez Garca Marina del Rosal Macas Technical Assistance: Mara Luisa Nogal Pars Mara Jos Bustos Snchez Esther Martn Forero Visiting Scientists: Mara Ballester Devis
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Redrejo-Rodrguez, M., Ishchenko, A. A., Saparbaev, M. K., Salas, M. L. and Salas, J. (2009) African swine fever virus AP endonuclease is a redox-sensitive enzyme that repairs alkylating and oxidative damage to DNA. Virology 390, 102-109.
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Rodrguez, I., Nogal, M. L.,Redrejo-Rodrguez, M., Bustos, M. J.and Salas, M. L. (2009) The African swine fever virus virion membrane protein pE248R is required for virus infectivity and an early postentry event. J. Virol. 83, 12290-12300. Surez, C., Salas, M. L. and Rodrguez, J. M. (2010) African swine fever virus polyprotein pp62 is essential for viral core development. J. Virol. 84, 176-187. Surez, C., Gutirrez-Berzal, J., Andrs, G., Salas, M. L. and Rodrguez, J. M. (2010) The African swine fever virus protein p17 is essential for the progression of the viral membrane precursors towards icosahedral intermediates. J. Virol. 84, 7484-7499. Ballester, M., Galindo-Cardiel, I., Gallardo, C., Argilaguet, J. M., Segals, J., Rodrguez, J. M. and Rodrguez, F. (2010) Intranuclear detection of African swine fever virus DNA in several cell types from formalin-fixed and paraffin-embedded tissues using a new in situ hybridization protocol. J. Virol. Methods 168, 38-43.
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Patents
Home M. L. Salas y J. M. Rodrguez (CSIC), L. K. Dixon (IAH). (2010). Vacuna contra el virus de la peste porcina Africana basada en virus recombinantes deficientes en replicacin. N Solicitud: ES1641.748.
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Home Modesto Redrejo Rodrguez (2009). La endonucleasa AP del virus de la peste porcina Africana y el papel biolgico del sistema viral de reparacin del ADN. Universidad Autnoma de Madrid. Directores: Jos Salas Falgueras y Mara Luisa Salas Falgueras. Cristina Surez Garca (2009). Papel de la poliprotena pp62 y de la protena p17 en la morfognesis del virus de la peste porcina Africana. Universidad Autnoma de Madrid. Director: Javier M. Rodrguez.
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B14
New strategies for prevention and control of viral diseases: foot-and-mouth disease virus as a model
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Foot-and-mouth disease virus (FMDV) is one of the major concerns for animal health. It is also an interesting model system for understanding the interactions of a highly variable virus and its natural hosts and the implications of these interactions on disease control. We are working in the development of new FMDV marker vaccines (peptides and DNA vaccines) that can induce protective humoral and cellular immune responses, using as animal model an important natural host: the pig. Some of these strategies are also being applied to the development of new vaccines against classical swine fever (CSF). We are also analyzing the functional role of FMDV proteins on the internalization, the replication cycle and the mechanisms mediating the pathogenesis of FMDV and other related viruses causing vesicular diseases, such as swine vesicular disease virus (SVDV), and vesicular stomatitis virus (VSV) in cultured cells and in animal models. Special attention is being paid to the functional implications of non-structural proteins, like those from the FMDV 3AB region, in virus virulence and host range. A parallel study of the functional implications of non-coding RNA regions is also being conducted. Besides providing basic information on the multiplication cycle of these viruses, the results obtained are being used for the identification of antiviral targets, attenuation determinants as well as the design of new vaccine strategies. As part of these studies, we are characterizing the recently identified inhibitory effect of valproic acid on the multiplication of enveloped viruses. Part of the work has been carried out in the BSL3 facilities at CISA-INIA.
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Figure 1. FMDV particles (red) entering IBRS-2 cells. Nuclei stained in blue.
Figure 2. Colocalization assays in IBRS-2 cells of SVDV (blue), transferrin (green) and tubulin (red).

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Home Group Leader: Francisco Sobrino Exit Scientific Staff: Margarita Siz Postdoctoral: Maria Flora Rosas Graduate Students: Ral Postigo Miguel ngel Martn Mara Teresa Snchez Miguel Rodrguez Yuri A. Vieira ngela Vzquez Flavia Caridi Undergraduate Students: Susan Realgen Abraham Arrizon Maria Rodolis Jackob Zimmermann Technical Assistance: Mnica Gonzlez Visiting Scientist: Beln Borrego (CISA-INIA)
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Publications
Rodrguez-Pulido, M., Sobrino, F., Borrego, B., Siz, M. (2009) RNA. Attenuated foot-and-mouth disease virus RNA carrying a deletion in the 3 non-coding region can elicit immunity in swine J. Virol.. 83, 3475-3485.
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Martn-Acebes, M.A., Gonzlez-Magaldi, M., Vzquez-Calvo, A., Armas-Portela, R. and Sobrino, F. (2009) Internalization of swine vesicular disease virus into cultured cells: a comparative study with foot-and-mouth disease virus. J. Virol. 83, 4216-4266 Sanchez-Aparicio, M.T., Rosas,M-F., Ferraz, R.M., Delgui, L., Veloso, J.J., Blanco, E, Villaverde, A. and Sobrino, F. (2009) Discriminating foot-and-mouth disease virus-infected and vaccinated animals by use of -galactosidase allosteric biosensors. Clin. Vaccine Immunol.16, 1228-1235 Martn-Acebes, MA., Rincn, V., Armas-Portela, R., Mateu, M.G. and Sobrino, F. (2010). A single amino acid substitution in the capsid of foot-and-mouth disease virus can increase acid-lability and confer resistance to acid-dependent uncoating inhibition. J. Virol. 84, 2902-2912 Rodrguez-Pulido, M., Sobrino, F., Borrego, B. and Siz, M. (2010). RNA immunization can protect mice against foot-and-mouth disease virus. Antiviral Res. 85, 556558. Martn-Acebes, M.A., Herrera, M., Domingo, E. and Sobrino, F. (2010). Cell density-dependent expression of viral antigens during persistence of foot-and-mouth disease virus in cell culture. Virology 403, 4755.
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Construccin peptdica dendrimrica para la prevencin de la fiebre aftosa en animales Inventores: C. Cubillo, E. Blanco, J. Brcena, F. Sobrino y D. Andreu. N de solicitud: P 200602142. Fecha de prioridad: 2-08-2006. Solicitud PCT (08/2007) N PCT ES 2007/070146. Uso del cido valproico como antiviral contra virus con envoltura. A. Vazquez, F. Sobrino, M.A. Martn y J.C. Siz. No Solicitud: P 200602142 (29 marzo 2010).
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Miguel ngel Martn Acebes (2009). Mecanismos de entrada y de organizacin del complejo de replicacin del virus de la fiebre aftosa: estudio comparativo con el virus de la enfermedad vesicular del cerdo y el virus de la estomatitis vesicular. Universidad Autnoma de Madrid. Co-dirigida por F. Sobrino y R. Armas. Miguel Ramn Rodrguez Pulido. Julio 2009. Estructura y funcin de la regin 3 no codificante del virus de la fiebre aftosa. Aplicacin a nuevas estrategias vacunales basadas en RNA. Universidad Autnoma de Madrid. Director: M. Siz. Ral Postigo Fernndez. Octubre 2009. Anlisis funcional de la protena 3A del virus de la fiebre aftosa. Universidad Autnoma de Madrid. Co-dirigida por F. Sobrino y M.F. Rosas. Mara Teresa Snchez Aparicio. Abril 2010. Estudio de protenas no estructurales del virus de la fiebre aftosa: anlisis funcionales y aplicacin al diagnstico viral. Co-dirigida por F. Sobrino y M.F. Rosas. Universidad Autnoma de Madrid.
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03B Virology and Microbiology

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Virology and Microbiology

Virology and Microbiology

Virology and Microbiology

Viral modulation of the immune response

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Virology and Microbiology

Viral modulation of the immune response

Figure 1. Mimicry of cytokines and cytokine receptors by herpesviruses and poxviruses.

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Virology and Microbiology

Viral modulation of the immune response

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Virology and Microbiology

Viral modulation of the immune response

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Virology and Microbiology

Viral modulation of the immune response

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Virology and Microbiology

Molecular bases of viral pathogenesis and anti-cancer potential

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Virology and Microbiology

Molecular bases of viral pathogenesis and anti-cancer potential

Research Summary Staff Publications Doctoral Theses

Virology and Microbiology

Molecular bases of viral pathogenesis and anti-cancer potential

Research Summary Staff Publications Doctoral Theses

Virology and Microbiology

Molecular bases of viral pathogenesis and anti-cancer potential

Research Summary Staff Publications Doctoral Theses

Virology and Microbiology

Molecular bases of viral pathogenesis and anti-cancer potential

Research Summary Staff Publications Doctoral Theses

Virology and Microbiology

Molecular Ecology of Extreme Environments

Research Summary Staff Publications Patents Doctoral Theses

Virology and Microbiology

Molecular Ecology of Extreme Environments

Research Summary Staff Publications Patents Doctoral Theses

Figura 1. Specific Cr(III) sequestering acidophilic fungi.

Figura 2. Acidophilic photosynthetic biofilm.

Virology and Microbiology

Molecular Ecology of Extreme Environments

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Virology and Microbiology

Molecular Ecology of Extreme Environments

Research Summary Staff Publications Patents Doctoral Theses

Virology and Microbiology

Molecular Ecology of Extreme Environments

Research Summary Staff Publications Patents Doctoral Theses

Virology and Microbiology

Molecular Ecology of Extreme Environments

Research Summary Staff Publications Patents Doctoral Theses

Virology and Microbiology

Molecular Ecology of Extreme Environments

Research Summary Staff Publications Patents Doctoral Theses

Virology and Microbiology

Bacterial Cell Division and Antibiotics Resistance

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Virology and Microbiology

Bacterial Cell Division and Antibiotics Resistance

Research Summary Staff Publications Other Activities

Virology and Microbiology