Cabecera CBMSO CSIC UAM

Thursday, 5th December 2019

Ultrahigh-throughput discovery and engineering of enzymes for biotechnological applications

 


Aurelio Hidalgo

BSciStaff

BPublications

 

Research summary:

Microbial diversity is a vast reservoir of genetic information that can be valorized through industrial application, from biosynthetic gene clusters to novel protein catalysts. The synergy between new experimental discovery tools based on biology and those based on nanotechnologies are instrumental to find relevant genes faster and more efficiently, enabling especially academic labs to undertake screening campaigns until now costly and limited to large enterprises.

 

En nuestro laboratorio utilizamos selecciones biológicas o métodos de cribado de ultra-alta capacidad basados en microfluídica para encontrar genes y enzimas de interés en la diversidad natural o artificial. Las selecciones biológicas acoplan la mejora en una propiedad de interés a la supervivencia de un huésped bajo presión de selección. Utilizando este y otros métodos de ingeniería de proteínas hemos desarrollado variantes estables y solubles de enzimas para biocatálisis, como esterasas, deshalogenasas, además de proteínas fluorescentes para aplicaciones de localización in vivo a altas temperaturas.

 

However, the complexity of cellular metabolism limits the application of biological selections. Microfluidics enables the miniaturization of assays with throughput of kHz and a 1000x reduction in volume and costs. Therefore, droplet microfluidics achieves throughputs typical of biological selections with none of their complexity. Within the H2020 project MetaFluidics, in our laboratory, we have implemented a fluorescence-based microfluidic sorting platform and assays to find stable hydrolases in thermal environments as well as other relevant enzymes for biocatalysis. This has entailed developing news hosts and methods for functional expression, compatible with enzymatic assays at high temperatures.

 

figura 1

Ultrahigh-thropughput screening using droplet microfluidics. A) Growth of microorganisms and cells in droplets after 24 h incubation. B) Ultrahigh-throughput screening of thermozymes using Thermus thermophilus as host. C) In vitro transcription translation (IVVTT) using T. thermophilus cell-free extracts. D) Digital PCR to detect rare microbes (mcirobial dark matter) and genes). Images by M. Almendros, A. Lopes and M. Sánchez

 


 

Funding:

  • Desarrollo de sistemas para la selección de proteínas termoestables mediante expresión in vivo e in vitro a alta temperatura. Entidad financiadora: UAM. UAM/89. Período: 01/02/2017-31/12/2021. Tipo de participación: IP.
  • Advanced toolbox for rapid and cost-effective functional metagenomic screening: microbiology meets microfluidics (METAFLUIDICS). Entidad financiadora: Unión Europea H2020. GA 685474. Período: 01/06/2016-30/05/2020. Tipo de participación: Coordinación.
  • Sustainable industrial processes based on a C-C bond-forming enzyme platform (CarbaZymes). Entidad financiadora: Unión Europea H2020. GA 635595. Período: 01/04/2015-31/03/2019. Tipo de participación: Socio.
  • Ultrahigh-throughput platform for the screening of thermostable proteins by thermophilic in vitro transcription-translation and microfluidics (HOTDROPS). Entidad financiadora: Unión Europea FP7. GA 324439. Período: 01/06/2013-30/05/2017. Tipo de participación: Socio.
  • Selección de alta eficiencia de biocatalizadores termoestables para química verde empleando microorganismos termófilos modificados. Entidad financiadora: Ministerio de Economía y Competitividad. Período: 01/01/2013-31/12/2016. Tipo de participación: Co-IP.

 


 

Last Doctoral Theses:

  • Yamal Al-ramahi González (2013) Fluorescent protein engineering and applications in thermophile microorganisms. Universidad Autónoma de Madrid. Supervisors: Aurelio Hidalgo y José Berenguer.
  • Noé R. Rivera (2013) Thermal stabilization of biotechnologically relevant proteins. Universidad Autónoma de Madrid. Supervisors: Aurelio Hidalgo y José Berenguer.

 


 

Ongoing Doctoral Theses:

  • Mercedes Sánchez Costa. Discovery and characterization of thermostable enzymes for biocatalysis. Supervisors: Aurelio Hidalgo y José Berenguer.
  • Sandra Bosch Reñé. Selection of thermostable biocatalysts for green chemistry using thermophile microorganisms. Supervisor: Aurelio Hidalgo.
  • Jorge Bravo Villanueva. Ultrahigh-throughput screening methods based on components from thermophile microorganisms. Supervisor: Aurelio Hidalgo.

 


 

Publications:

  • J. Castillo, S. Caminata Landriel, M. Sánchez Costa, O. A. Taboga, J. Berenguer, A. Hidalgo, S. A. Ferrarotti, H. Costa, Protein Eng., Des. Sel. 2018, 76, 224.
  • T. Consolati, J. M. Bolivar, Z. Petrasek, J. Berenguer, A. Hidalgo, J. M. Guisan, B. Nidetzky, ACS Appl. Mater. Interfaces 2018, acsami.7b16639.
  • A. L. Ribeiro, M. Mencía, A. Hidalgo, Methods Mol. Biol. 2018, 1685, 131–143.
  • J. Berenguer, M. Mencía, A. Hidalgo, Microbial Biotechnology 2017, 10, 46–49.
  • A. L. Ribeiro, M. Sánchez, A. Hidalgo, J. Berenguer, Methods Mol. Biol. 2017, 1645, 297–312.
  • A. Vettone, M. Serpe, A. Hidalgo, J. Berenguer, G. Del Monaco, A. Valenti, M. Rossi, M. Ciaramella, G. Perugino, Extremophiles 2016, 20, 1–13.
  • A. Hidalgo, A. Schließmann, U. T. Bornscheuer, Methods Mol. Biol. 2014, 1179, 207–212.
  • L. L. Torres, A. Cantero, M. Del Valle, A. Marina, F. Lopez-Gallego, J. M. Guisan, J. Berenguer, A. Hidalgo, Appl. Environ. Microbiol. 2013, 79, 1555–1562.
  • A. Hidalgo, J. Berenguer, Current Biotechnology 2013, 2, 304–312.
  • L. L. Torres, E. R. Ferreras, A. Cantero, A. Hidalgo, J. Berenguer, Microb Cell Fact 2012, 11, 105.
  • L. L. Torres, A. Schließmann, M. Schmidt, N. Silva-Martin, J. A. Hermoso, J. Berenguer, U. T. Bornscheuer, A. Hidalgo, Org. Biomol. Chem. 2012, 10, 3388–3392.

Human immunodeficiency virus reverse transcriptase and antiretroviral therapy

 

 2019 07 03 Grupo Luis Menéndez 400px

 


Luis Menéndez-Arias

 

 

BSciStaff

 

BPublications

 

 

Research summary

Infections caused by human immunodeficiency viruses type 1 and type 2 (HIV-1 and HIV-2, respectively) constitute a major burden to human health worldwide. Despite significant advances in antiretroviral therapy HIV still causes 1.6 million deaths each year. The HIV genome is composed of two copies of single-stranded RNA. The viral reverse transcriptase (RT) is responsible for the replication of the HIV genome. RT inhibitors constitute the backbone of the most popular and effective therapies.

For years, our efforts have been directed towards achieving two major goals: (1) the elucidation of molecular mechanisms involved in RT inhibitor resistance; and (2) understanding the role of different amino acids in the nucleotide specificity of HIV-1 and HIV-2 RTs, as well as in their fidelity of DNA synthesis. We have engineered HIV-1 RT variants with increased thermal stability and high fidelity that are currently marketed as biotechnological tools for many applications, covering basic and translational research. Future challenges involve the development of RTs with high nucleic acid binding affinity suitable for RNA amplification from single cells.

HIV-2 shows natural resistance to nonnucleoside RT inhibitors (NNRTIs) and several protease inhibitors. Nucleoside RT inhibitors (NRTIs) constitute the backbone of therapies against HIV. However, HIV-1 and HIV-2 show different mutational pathways of NRTI resistance. We have recently demonstrated that HIV-2 RT residues Met-73 and Ile-75 restrict the development of M41L, D67N, K70R and T215Y (known as thymidine analogue resistance mutations). The reported findings explain why thymidine analogue resistance mutations are rarely observed in treated HIV-2-infected patients.

On the other hand, prevalence of drug-resistant HIV strains is increasing in less developed countries. Therefore, we anticipate an increasing interest in unexploited targets of antiretroviral intervention. In this context, RNase H activity and inhibition, as well as host factors that could eventually block HIV-1 replication are also important topics of research in our laboratory.

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Comparison of the structural models of wild-type and mutant HIV-1 RTs D67N/K70R and D67N/K70R/K73M, complexed with double-stranded DNA and an incoming dNTP. View of the β3-β4 hairpin loop, the incoming dNTP and the two Mg2+ ions, highlighting the effects of Met-73 on the interaction of Arg-70 with the dNTP. For details see Álvarez et al. (2018) J. Biol. Chem., 293, 2247-2259.
        RT-PCR assays showing the effect of the temperature on the cDNA synthesis catalyzed by engineered recombinant HIV-1 group O RT variants (Matamoros et al. 2013; Biochemistry 52, 9318).

 


 

Relevant publications:

  • Menéndez-Arias, L. (2013) Molecular basis of human immunodeficiency virus type 1 drug resistance: Overview and recent developments. Antiviral Res. 98, 93-120; doi: 10.1016/j.an-tiviral.2013.01.007.
  • Betancor, G., Álvarez, M., Marcelli, B., Andrés, C., Martínez, M.A., Menéndez-Arias, L. (2015) Effects of HIV-1 reverse transcriptase connection subdomain mutations on polypurine tract removal and initiation of (+)-strand DNA synthesis. Nucleic Acids Res. 43, 2259-2270.   
  • Álvarez, M., Nevot, M., Mendieta, J., Martínez, M.A., Menéndez-Arias, L. (2018) Amino acid residues in HIV-2 reverse transcriptase that restrict the development of nucleoside analogue resistance through the excision pathway. J. Biol. Chem. 293, 2247-2259; doi:10.1074/jbc.RA117.000177.
  • Sebastián-Martín, A., Barrioluengo, V., Menéndez-Arias, L. (2018) Transcriptional inaccuracy threshold attenuates differences in RNA-dependent DNA synthesis fidelity between retroviral reverse transcriptases. Scientific Reports 8, 627; doi: 10.1038/s41598-017-18974-8.
  • Luczkowiak, J., Matamoros, T., Menéndez-Arias, L. (2018) Template-primer binding affinity and RNase H cleavage specificity contribute to the strand transfer efficiency of HIV-1 reverse transcriptase. J. Biol. Chem. 293, 13351-13363; doi:10.1074/jbc.RA118.004324.

 

Yeast enzymes bioengineering to generate bioactive compounds

 

GrupoLobato 400px
 


María Fernández Lobato

BSciStaff

BPublications

 

Research summary:

We work with microorganisms of biotechnological interest, mainly fungi and yeasts, producers of bioactive compounds. We try to connect the generation of knowledge to the development of biotechnological applications. Basically we focus on the characterization of new enzymes producing bioactive compounds, the analysis of their structural-functional determinants, the operational improvement using molecular biology tools and in obtaining and characterization of new molecules with potential industrial utility. We have patented in different countries the industrial applicability of most proteins characterized and designed methods for their attachment to solid supports.

Fig1 300px   Fig 02 300px
The Xd-INV from Xanthophyllomyces dendrorhous 3D-Structure, a fructofuranosidase producing the prebiotic neokestose. Close-up view of active site including neo-erlose (fructosil maltose).

 

The conformation of the substrates at the Ffase active site. A. Six units of inulin (left) and fructosylnystose (right) molecules represented as spheres. B. Inulin (brown) and fructosylnystose (lime green) moieties in stick representation are superimposed.

 

During the last years we have been characterizing and studying several non-conventional yeast proteins (from genera Xanthophyllomyces, Schwanniomyces, Rhodotorula, etc.) showing glycosyltransferase activity, and applicable in the production of sugars with prebiotic properties. All are glycosylhydrolases (GH) structurally included in family GH32, 31, 13 or 2. Indeed, we have resolved the 3-D structure of the first yeast protein including in family GH32, assigned a function to the beta-sandwich domain that is present in all members of this family and proved that the oligomerization is directly involved in the substrate recognition and specificity. We have improved enzymes and altered their biosynthetic activity using molecular bioengineering tecniques.  More information:

Glicoenz200

   INMARE

 


 

Selected publications:

  • Ramírez-Escudero, M., M. Gimeno-Pérez, B. González, D. Linde, Z. Merdzo, M. Fernández-Lobato* and J. Sanz-Aparicio* (2016) Structural Analysis of β-Fructofuranosidase from Xanthophyllomyces dendrorhous Reveals Unique Features and the Crucial Role of N-glycosylation in Oligomerization and Activity. J. Biol. Chem (doi/10.1074/jbc.M115.708495). *Both corresponding authors.
  • Gimeno-Pérez,M., D. Linde, L. Fernández-Arrojo, F. J. Plou, and M. Fernández Lobato (2015) Heterologous overproduction of β-fructofuranosidase from Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars. Appl Microbiol Biotechnol. 98 (8) 3459-3467 (doi: 10.1007/s00253-014-6145-1).
  • de Abreu, M.A., Alvaro-Benito, M., Plou, F.J., Fernández Lobato, M.* and Alcalde, M.* (2013) Synthesis of 6-kestose using a highly efficient β-fructofuranosidase engineered by directed evolution. Adv. Synth. Catal. 355 (9), 1698-1702. * Both corresponding authors. (doi: 10.1002/adsc.201200769).
  • Linde, D., Estévez, M., Plou, F. J. and Fernández Lobato, M. (2012) Analysis of the neofructooligosaccharides production mediated by the extracellular β-fructofuranosidase Xd-INV from Xanthophyllomyces dendrorhous. Bioresour. Technol. 109, 123-130 (doi: 10.1016/j.biortech.2012.01.023)
  • Alvaro-Benito, M., Sainz-Polo, M.A., González, B., Plou, F.J., Fernández-Lobato*, M. and Sanz-Aparicio*, J. (2012) Structural and kinetic insight reveal that the amino acid pair Gln-228/Asn-254 modulates the transfructosylating specificity of Schwanniomyces occidentalis β-fructofuranosidase, and enzyme that produces prebiotics. J. Biol. Chem. 287, 19674-19686. *Both corresponding authors. (doi:10.1074/jbc.M112.355503)

 

Selected patents:

P200402994. New enzyme for the prebiotic production: WO 2006/064078 A1; PCT-ES2005/070177; Europa No 05825223.0. Primer premio a la mejor patente Madrid+d 2008.

P200501875. New fructofuranosydase for prebiotic oligosaccharides obtaining: PCT-ES2006/000435. Europa Nº 06807883.1; USA No. 11/997.233

P200503195. "New fructofuranosydase for the 6-kestose production: PCT-ES2006/000693; Europea No. 06841745-0; USA No. 12/159.164; Japón No. 2008-547993.

P200930001. Biocatalizador inmovilizado basado en alginato para la biotransformación de carbohidratos: PCT-ES2010/070104


 

Selected Doctoral Theses:

  • Patricia Gutiérrez Alonso (2013) Caracterización de dos glicosiltransferasas de oligosacáridos prebióticos de las levaduras Phaffia rhodozyma y Rhodotorula dairenensis. UAM.
  • Miguel Antonio de Abreu Felipe (2011) Studies aimed at improving the funtionality of non-conventional yeast enzymes able to synthesize prebiotic oligosaccarides. UAM. European Program
  • Miguel Álvaro Benito (2011) The study of β-fructofuranosidase from Schwanniomyces occidentalis reveals new functional elements in the family GH32 of glycosyltransferases and an unconventional genetic code use in this yeast. UAM. European Program
  • María Dolores Linde López (2010) Caracterización Bioquímica, molecular y estructural de una β-fructofuranosidasa con capacidad transferasa de la levadura Phaffia rhodozyma aplicable a la producción de oligosacáridos prebióticos. UAM

 

Images gallery:

Otrosgrupos22mimi Otrosgrupos00-200 Otrosgrupos01-200 Otrosgrupos02-200
Otrosgrupos03-300 Otrosgrupos04-300 Otrosgrupos05-200  

 

 

 

New strategies for prevention and control of viral diseases: foot-and-mouth disease virus as a model

 grupo400

 


Francisco Sobrino

BSciStaff

BPublications

 

Research summary:

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 peptide marker vaccines that can induce protective humoral and cellular immune responses, using pig and cattle, important domestics hosts, as animal models. We have also analyzed the functional role of FMDV proteins on the viral particle stability and 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). Special attention has been paid to the functional implications of nonstructural proteins in virus virulence and host range. The role of different cellular lipids in the multiplication of these and other viruses such as West Nile virus (WNV), responsible for an important zoonosis, have also been addressed. As part of these studies, we have continued collaborating in the characterization of the inhibitory effect of valproic acid and other antiviral compounds targeting cellular metabolism such as lauryl gallate on the multiplication of FMDV and of other enveloped viruses, like African swine fever virus (ASFV) and type I herpesvirus.

 

figure 1

 

In a second line of research, Margarita Sáiz undertakes the analysis of the interplay between FMDV and the innate immunity system. With the aim of unveiling the molecular mechanisms exerted by FMDV for immune evasion, the role of the two virally-encoded proteases is being studied. We have shown that the FMDV Leader protease targets innate immune sensor LGP2 for cleavage, resulting in lower levels of IFN-β and antiviral activity. The interference of the proteases with different signaling routes triggered by viral sensors and their interaction with a variety of immune effectors as potential targets for viral antagonism are being analyzed.

 

figure 2

 

On the other hand, the biotherapeutic applications of synthetic non-coding RNAs derived from the viral genome, and known to elicit a broad spectrum antiviral activity, is being explored. We have shown that RNA delivery enhanced the specific B- and T-cell mediated immune responses elicited by a conventional FMD vaccine, increasing the rate of protection against viral challenge. The activity of these molecules in livestock species is being analyzed to assess their biotechnological potential for the development of new antiviral molecules and vaccine adjuvants.

 


 

Relevant publications:

  • Borrego, B., Rodríguez-Pulido, B., Mateos, F., de la Losa, N., Sobrino, F. and Sáiz*, M. Delivery of synthetic RNA can enhance the immunogenicity of vaccines against foot-and-mouth disease virus (FMDV) in mice. Vaccine. 40, 4375-4381 (2013).
  • Sanchez-Aparicio, M.T., Rosas, M.F. and Sobrino, F*. Characterization of a nuclear localization signal in the foot-and-mouth disease virus polymerase. Virology 444, 203-210 (2013).
  • Blanco, E., Cubillos, C., Moreno, N., Bárcena, J., de la Torre, B.G., Andreu and Sobrino, F*. B epitope multiplicity/ and B/T epitope orientation influence immunogenicity of foot-and-mouth disease peptide vaccines. Clin. Dev. Immunol. 2013:475960 (2013).
  • Vazquez-Calvo, A. Caridi, F. Sobrino*, F. and Martín-Acebes, M.A. An increase in acid resistance of foot-and-mouth disease virus capsid is mediated by a tyrosine substitution of the VP2 histidine previously associated with VP0 cleavage. J. Virol. 88, 3039-3042 (2014).
  • Martín-Acebes, M.A., Merino-Ramos, T., Blázquez, A-B., Casas, J., Escribano-Romero, E., Sobrino, F*. and Saiz, J.C*. The Composition of West Nile virus Lipid Envelope Unveils a Role of Sphingolipid Metabolism on Flavivirus Biogenesis J. Virol. 88(20), 12041-54 (2014).
  • González-Magaldi, M., Martín-Acebes, M., Kremer, L. and Sobrino, F*. Membrane topology and cellular dynamics of foot-and-mouth disease virus 3A protein. PLoS ONE. 9(10): e106685. (2014).
  • Caridi, F., Vázquez-Calvo, A., Sobrino, F*. and Martín-Acebes, M.A. The pH stability of foot-and-mouth disease virus particles is modulated by residues Located at the pentameric interface and in the N terminus of VP3. J. Virol. (2015).
  • Borrego, B., Rodríguez-Pulido, M., Revilla, C., Álvarez, B., Sobrino F., Domínguez, J. and Sáiz, M*. Synthetic RNAs mimicking structural domains in the foot-and-mouth disease virus (FMDV) genome elicit a broad innate immune response in porcine cells triggered by RIG-I and TLR activation. Viruses 7, 3954-3973. doi:10.3390/v7072807 (2015).
  • Martin-Acebes, M.A., Gabandé-Rodríguez, E., García-Cabrero, A.M., Sánchez, M.P., Ledesma, M.D., Sobrino, F*. and Saiz, J.C.* Host Sphingomyelin Modulates West Nile Virus Infection in vivo. J. Lipid. Res. 57, 422-32. doi: 10.1194/jlr.M064212 (2016).
  • Blanco E.*, Guerra, G., de la Torre, B.G., Defaus, S., Andreu, D*. and Sobrino, F*. Full protection of swine against foot-and-mouth disease challenge by a bivalent B-cell epitope dendrimer peptide. Antiviral Res. 129, 74-80. doi: 10.1016/j.antiviral.2016.03.005 (2016).
  • The amino acid substitution Q65H in the 2C protein of swine vesicular disease virus confers resistance to Golgi disrupting drugs. Vázquez-Calvo, A., Caridi, F., González-Magaldi, M., Saiz, J.C. Sobrino, F*. and Martín-Acebes, M.A.*. Frontiers Microbiol. 7:612. doi: 10.3389/fmicb.2016.00612 (2016).
  • Borrego, B., Blanco, E., Rodríguez Pulido, M., Mateos, F., Lorenzo, G., Cardillo, S., Smitsaart, E., Sobrino, F. and Sáiz, M. (2017) Combined administration of synthetic RNA and a conventional vaccine improves immune responses and protection against foot-and-mouth disease virus in swine. Antiviral Res. 142, 30-36.doi: 10.1016/j.antiviral.2017.03.009.
  • Caridi, F., Vázquez-Calvo, A., Borrego, B., McCullough, K., Summerfield, A., Sobrino, F. and Martín-Acebes, M.A. (F. Sobrino, corresponding author) (2017) Preserved immunogenicity of an inactivated vaccine based on foot-and-mouth disease virus particles with improved stability. Vet. Microbiol. 203, 275 - 279.doi: 10.1016/j.vetmic.2017.03.
  • Bohórquez, J.A., Defaus, S., Muñoz-González, S., Perez-Simó, M., Rosell, R., Fraile, L., Sobrino, F., Andreu, D. and Ganges, L. (2017) A bivalent dendrimeric peptide bearing a T-cell epitope from foot-and-mouth disease virus protein 3A improves humoral response against classical swine fever virus. Virus Res. 238, 8-12.doi: 10.1016/j.virusres.2017.05.020.
  • Soria, I., Quattrocchi, V., Langellotti, C., Gammella, M., Digiacomo, S., Garcia de la Torre. B., Andreu, D., Montoya, M., Sobrino, F., Blanco, E. and Zamorano, P. (F. Sobrino, corresponding author). (2017) Dendrimeric peptides can confer protection against foot-and-mouth disease virus in cattle. PLoS ONE 12 (9): e0185184.doi: 10.1371/journal.pone.0185184.
  • Blanco, E., Andreu, D. and Sobrino, F. (2017) Peptide vaccines against foot-and-mouth disease virus. In Sobrino, F. and Domigo, E. (eds.) Foot and mouth disease virus: current research and emerging trends. Caister Academic Press, Norfolk, UK. pp.317-332.doi: 10.21775/9781910190517.15.
  • Rodríguez Pulido, M. and Sáiz, M. (2017) Molecular mechanisms of foot-and-mouth disease virus targeting the host antiviral response. Front. Cell .Infect. Microbiol. 7:252.doi: 10.3389/fcimb.2017.00252.
  • Soria, I., Quattrocchi, V., Langellotti, C., Pérez Filgueira, M., Romera, S., Schammas, J., Buscafuco, D., Garcia de la Torre, B., Andreu, D., Sobrino, F., Blanco, E. and Zamorano, P. (F. Sobrino, corresponding author) (2018) Immune response and partial protection against heterologous foot-and-mouth disease virus induced by dendrimer peptides in cattle. J. Immunol. Res.doi:10.1155/2018/3497401.
  • Rodríguez Pulido, M., del Amo, L., Sobrino, F. and Sáiz, M. (2018) Synthetic RNA derived from the foot-and-mouth disease virus genome elicits antiviral responses in bovine and porcine cells through IRF3 activation. Vet. Microbiol. 221, 8-12.doi: 10.1016/j.vetmic.2018.05.015.
  • Rodríguez Pulido, M., Sánchez-Aparicio, M.T., Martínez-Salas, E., García-Sastre, A., Sobrino, F. and Sáiz, M. (2018) Innate immune sensor LGP2 is cleaved by the leader protease of foot-and-mouth disease virus. PLoS Pathog. 14(6):e1007135.doi: 10.1371/journal.ppat.1007135.
  • de la Higuera, I., Ferrer-Orta, C.; Moreno, E., De Ávila, A., Soria, M.E., Kamlendra, S., Caridi, F., Sobrino, F., Sarafianos, C., Perales, C., Verdaguer, N. and Domingo, E. (2018) Contribution of a multifunctional polymerase region of foot-and-mouth disease virus to lethal mutagenesis. J. Virol. 92:e01119-18.doi: 10.1128/JVI.01119-18.

* Corresponding author

 


 

Edited books:

  • Sobrino, F. and Domingo, E. (eds) (2017). Foot and mouth disease virus: current research and emerging trends. Caister Academic Press, Norfolk, UK. ISBN: 978-1-910190-51-7 (paperback); 978-1-910190-52-4 (ebook).

 


 

Patents:

  • Vacunas peptídicas para la prevención de la fiebre aftosa. David Andreu, Juan Bárcena del Riego, Esther Blanco, Carolina Cubillos, Beatriz García de la Torre, Marta Monsó, Francisco Sobrino. P20130101063. Argentina (03/04/2013).
  • Peptide vaccines for the prevention of foot-and-mouth disease. David Andreu, Juan Bárcena del Riego, Esther Blanco, Carolina Cubillos, Beatriz García de la Torre, Marta Monsó, Francisco Sobrino.P. number: 2013800289123. P.R.China (01/12/2014).
  • Use of esters derived from gallic acid as antivirals. P. de León, A. L. Carrascosa, M.J. Bustos, F. Sobrino. E. Torres, R. Cañas. Patente ES1641.1421 (12-11-2018).

 


 

Doctoral Theses:

  • Yuri A. Vieira (2015). Contribuciones al estudio de la funcionalidad de las proteínas no estructurales del virus de la fiebre aftosa. Universidad Autónoma de Madrid. Directores: M.F. Rosas y F. Sobrino.
  • Flavia Caridi (2017). Bases Moleculares de la estabilidad de la cápsida de los aftovirus. Universidad Autónoma de Madrid. Directores: F. Sobrino y M.A. Martín Acebes. Sobresaliente “Cum Laude”.

Virology and Microbiology

                Bacterial morphogenesis

 

 

grupo-400

 


 

 

Miguel Angel de Pedro

BSciStaff

BPublications

 

 

 

 

Research summary:

Our research is focused on the study at the molecular and physiological levels of the cell wall (sacculus) as the primary morphogenetic element of the bacterial cell. We continued working out the mechanisms of synthesis and growth of the cell wall in polymorphic bacteria. In addition, in the last two years we made important efforts in two new directions: the characterization of the process of D-amino acid production and release by some bacterial species, and the study of the diversity and plasticity of the bacterial wall.

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"In vivo" visualization of cell wall biosynthetic sites in : A) Rhizobium meliloti, B) Roseobacter litoralis, C) Erythrobacter aquimaris and D) Asticaccaulis biprosthecium, with a fluorescent D-amino acid.

In the first case, we are studying the biochemistry and physiology of the process, as well as its biological meaning in natural environments and poly-microbial communities. This type of mechanism may play an important role in signaling and timing responses in communities were different bacterial species compete. We want to establish the dispersion of the established mechanism (release of D-amino acids) and look for similar mechanisms mediated by other types of effector molecules. In the second case, we are focusing our efforts on a better assessment of the structural and compositional diversity of bacterial cell walls (variability), and the adaptive responses of the cell wall to changing environmental conditions (plasticity), including pathological processes. Both aspects seem to be far more variable than previously suspected. An accurate knowledge of both would lead to a better understanding of bacterial evolution and adaptation, and could open new ways to control natural populations. For the immediate future we will continue our current research, and start development of new methods for a systematic and massive analysis of cell wall diversity and growth pattern.

 

 

 

 

 

 


 

Selected publications:

  • Cava, F., de Pedro, M.A., Lam, H., Davis, B.M., and Waldor, M.K. (2011) Distinct pathways for modification of the bacterial cell wall by non-canonical D-amino acids. EMBO J. 30, 3442-3453.
  • Cava, F., Lam, H., de Pedro, M.A., and Waldor, M.K. (2011) Emerging knowledge of regulatory roles of D-amino acids in bacteria. Cell. Mol. Life Sci. 68:817-831.
  • Slamti, L., de Pedro, M.A. Guichet, M., and Picardeau, M. (2011) Deciphering morphologycal determinants of the helix shaped Leptospira. J. Bacteriol. 193, 6266-6275.
  • González-Leiza, S.M., de Pedro, M.A., and Ayala, J.A. (2011) AmpH, a bifunctional DD-endopeptidase and DD-carboxypeptidase of Escherichia coli. J. Bacteriol. 193, 6887-6894.
  • Acosta, F., Alvarez, L., de Pedro, M.A., and Berenguer, J. (2012) Localized synthesis of the outer envelope from Thermus thermophilus. Extremophiles 16, 267-275

Effects of extrachromosomal elements on behavior of its host and mechanisms of horizontal gene transfer in Bacillus

 

grupo-400

 


 

 

Wilfried J.J Meijer

BSciStaff

BPublications

 

 

 

 

Research summary:

fig01-300
Inhibitory effect of pLS20-encoded protein Rok-LS20 on competence of B. subtilis. Competent cells are green due to expression of the green fluorescent protein engineered to be under the control of specific competence promoter. Membranes are stained red. The strain contains a copy of the pLS20-located gene rokLS20 under the control of an IPTG-inducible promoter. A and B. Cells without and with induction of Rok-LS20.

fig02-300

Genetic map of B. subtilis conjugative plasmid pLS20..

Mobile genetic elements (MGE), e.g. phages, plasmids, transposons and ICEs, can be transferred horizontally between cells affecting 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 dispersion of antibiotic resistance.
Little is known, especially in Gram-positive bacteria, about the transcriptional regulation of mobility genes or how MGE affects its host. A better understanding of these issues is warranted to face important threats, like antibiotic resistance. 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 easy amenable to genetic manipulation; and (iv) B. subtilis is related to pathogenic/fastidious bacteria like Bacillus anthracis, B. cereus and, although more distantly, to Listeria monocytogenes. So far, no sequence of conjugative B. subtilis plasmids was known. We have now sequenced and annotated the two large B. subtilis plasmids and are functionally analyzing them with the major aims to get insight in regulation of the mobility genes and effect on their host.
Many Gram positive bacteria with industrial or scientific importance are reluctant to genetic manipulation. Our goal is to construct versatile vectors allowing easy genetic manipulation of such bacteria based on the conjugation systems we study. For this we study several additional aspects of the conjugative plasmids.
Upon infection, phages often drastically alter the behaviour of B. subtilis. However, neither sequence nor mechanistic information of how these phages exert their effects is known. We are attempting to understand the mechanism underlying these alterations using two temperate phages as model systems.


 

Relevant publications:

  • Singh, P. K., Ramachandran, G., Durán-Alcalde, L., Alonso, C. Wu, L.J., and Meijer, W.J.J. (2012) Inhibition of Bacillus subtilis natural competence by a native, conjugative plasmid-encoded comK repressor protein. Environ. Microbiol. 14, 2812-25

Virus engineering and Nanobiotechnology

 

Mauricio GarciaMateuGrupo
 


Mauricio García Mateu

BSciStaff

BPublications

 

Research summary:

Major research goals: We use protein engineering techniques and biochemical, biophysical and virological analyses to study assembly, conformational stability and dynamics and physical properties of viruses, and their biological relevance (Mateu (ed.) (2013) Structure and Physics of Viruses, Springer 2013; Mateu (2013) Arch.Biochem.Biophys. 531,65-79). Based on these studies, we aim also at the design and analysis of genetically and/or structurally modified viral particles for the development of biomedical and bionanotechnological applications (Mateu (2016). In Protein-based Engineered Nanostructures, Springer 2016, pp.83-120).

Scientific relevance and technological implications: In-depth knowledge of certain key processes for viral infection, including virus morphogenesis, structural rearrangements and uncoating; application of this knowledge for the design of vaccines, antiviral drugs, biomaterials and modified nanoparticles for biomedical or bionanotechnological uses.

Some recent results: i) The combined use of atomic force microscopy (AFM) and electron microscopy allowed us to experimentally determine for the first time the reversible pathway and intermediates of assembly and disassembly of a structurally simple spherical virus (Fig.1). ii) Using mutational analysis and the determination of mechanical properties of virus particles by AFM we have discovered a relationship between genetic changes that alter the mechanical stiffness of virus particles and changes in the propensity of the latter to undergo conformational changes related to the infection process (Fig.2). iii) we have characterized the structure, dynamics and mechanical properties of a bidimensional nanocoating made by self-assembly of the HIV capsid protein on a solid matrix. These and other studies by our group have implications for a better understanding of processes essential for viral infection, the design of new antivirals that may inhibit these processes, and the development of nanoparticles and bidimensional biomaterials with improved mechanical properties for applications such as targeted drug delivery or tissue regeneration.

Mauricio GarciaMateuFig1 .............. Mauricio-GarciaMateuFig2.jpg
 

Intermediates during the reversible assembly-disassembly pathway of the minute virus of mice capsid. Assembly proceeds through formation of a nucleus and the gradual association of capsid building blocks formed by trimers of the capsid protein.

  

Linear relationship between an increase in mechanical stiffness of the minute virus of mice mediated by capsid-nucleic acid interactions, and an increase in its resistance against loss of infectivity by a heat-induced conformational change. This virus appears to have evolved genome binding sites in the capsid inner wall, which increases its stiffness and, in consequence, its resistance against thermal inactivation in the extracellular medium.





Selected publications (last 10 years):

  • Mateo, R., Luna, E., Rincón, V. and Mateu, M.G. (2008). Engineering viable foot-and-mouth disease virus of increased stability as a step in the development of improved vaccines. J.Virol. 82, 12232-12240.
  • Carrasco, C., Castellanos, M., de Pablo, P.J. and Mateu, M.G. (2008). Manipulation of the mechanical properties of a virus by protein engineering. Proc. Natl. Acad. Sci. USA 105, 4150-4155.
  • Bocanegra, R., Domenech, R., Nevot, M., Rodriguez-Huete, A., López, I., Fuertes, M.A., Cavasotto, C., Martínez, M.A., Neira, J.L. and Mateu, M.G. (2011). Rationally designed interfacial peptides are efficient in vitro inhibitors of HIV-1 capsid assembly with antiviral activity. PLoS ONE. 6, e23877.
  • Castellanos, M., Pérez, R., Carrasco, C., Hernando-Pérez, M., Gómez-Herrero, J., de Pablo, P.J. and Mateu, M.G. (2012). A balance between stiffness and elasticity provides a mechanical foundation for the infectivity of a virus. Proc. Natl. Acad. Sci. USA 109, 12028-12033.
  • Mateu, M.G. (ed.) (2013). Structure and Physics of Viruses. Springer, Dordrecht, The Netherlands.
  • Rincón, V., Rodriguez-Huete, A., López-Argüello, S., Ibarra-Molero, B., Sánchez-Ruiz, J.M., Harmsen, M.M. and Mateu, M.G. (2014). Identification of the structural basis of thermal lability of a virus provides a rationale for improved vaccines. Structure 22, 1560-1570.
  • Castellanos, M., Carrillo, P.J.P. and Mateu, M.G. (2015). Quantitatively probing propensity for structural transitions in engineered virus nanoparticles by single molecule mechanical analysis. Nanoscale 7, 5654-5664.
  • Valbuena, A. and Mateu, M.G. (2015). Quantification and modification of the equlibrium dynamics and mechanics of a virus capsid lattice self-assembled as a protein nanocoating. Nanoscale 7, 14953-14964.
  • Medrano, M., Fuertes, M.A., Valbuena, A., Carrillo, P.J.P., Rodríguez-Huete, A. and Mateu, M.G. (2016). Imaging and quantitation of a succession of transient intermediates reveal the self-assembly pathway of a simle icosahedral virus capsid. J. Am. Chem. Soc. 138, 15385-15396.
  • Carrillo, P.J.P., Medrano, M., Valbuena, A., Rodríguez-Huete, A., Castellanos, M., Pérez, R. and Mateu, M.G. (2017). Amino acid side chains buried along intersubunit interfaces in a viral capsid preserve low mechanical stiffness associated with virus infectivity. ACS Nano 11, 2194-2208.
  • Valbuena, A., Rodríguez-Huete, A. and Mateu, M.G. (2018). Mechanical stiffening of human rhinovirus by cavity-fiilling antiviral drugs. Nanoscale 10, 1440-1452.

Last doctoral theses:

Milagros Castellanos Molina (2011). Análisis mutacional de propiedades estructurales y mecánicas del virus diminuto del ratón, y de sus implicaciones biológicas. Universidad Autónoma de Madrid. Director: Mauricio G. Mateu.

Rebeca Bocanegra Rojo (2011). Ensamblaje in vitro de la cápsida del virus de la inmunodeficiencia humana, y su inhibición por péptidos diseñados racionalmente. Universidad Autónoma de Madrid. Director: Mauricio G. Mateu.

Verónica Rincón Forero (2012). Relaciones estructura-función en la cápsida del virus de la fiebre aftosa: algunas implicaciones para el desarrollo de vacunas y antivirales. Universidad Autónoma de Madrid. Director: Mauricio G. Mateu.

Verónica Rincón Forero (2012). Relaciones estructura-función en la cápsida del virus de la fiebre aftosa: algunas implicaciones para el desarrollo de vacunas y antivirales. Universidad Autónoma de Madrid. Director: Mauricio G. Mateu.

Pablo Pérez Carrillo (2015). Papel de Interfases y residuos interfásicos en la elasticidad mecánica y dinámica conformacional de partículas del virus diminuto del ratón. Universidad Autónoma de Madrid. Director: Mauricio G. Mateu.

María Medrano García (2018). Fundamentos moleculares del ensamblaje y las propiedades mecánicas de partículas víricas con aplicaciones biotecnológicas y nanotecnológicas. Universidad Autónoma de Madrid. Director: Mauricio G. Mateu. (to be defended in April 2018)


 

Genetic variability of RNA viruses

 grupo400

 


Esteban Domingo Solans

BSciStaff

BPublications

 

Research summary:

Our group pioneered (1978-1990) the discovery of viral quasispecies, a highly complex and dynamic population structure, with profound implications in viral evolution and pathogenesis. Our results of the last 40 years on quasispecies dynamics have been amply confirmed by application of deep sequencing methodology in several laboratories in the last three years. The genome of an RNA viral population is a weighted average of myriads of different, related sequences which are changing continuously. The consensus (or average) sequence that we write for convenience, and that fills textbooks and data banks, is an abstraction that may not even exist in the population that the sequence aims at representing. Dynamic mutant clouds are prepared to respond to changing environments by selection of subsets of sequences preferentially over others, an event crucial to disease progression.

 

Quasispecies dynamics demands that new approaches be investigated for the prevention and treatment of diseases associated with RNA viruses, to counteract the adaptive capacity conferred by the mutant clouds.

 

We are working in the development of new antiviral strategies that can avoid selection of treatment-escape mutant viruses. The work involves mainly three viruses: the picornavirus foot-and-mouth disease virus (FMDV), the arenavirus lymphocytic choriomeningitis virus (LCMV), and more recently the hepacivirus hepatitis C virus (HCV). We are collaborating with several groups to expand the scope of our work: with the group of Nuria Verdaguer (IBM, CSIC) on studies of structural alterations of the viral polymerase of mutagen-resistant mutants of FMDV; with Juan Carlos de la Torre (Scripps Research Institute, La Jolla) on lethal mutagenesis of LCMV; with Pablo Gastaminza (CNB, CSIC), Celia Perales (IIS, Fundación Jiménez Díaz), Josep Quer (Hospital Vall d’Hebrón), and Aurora Sánchez (IIB, CSIC-UAM) on HCV dynamics and host-virus interactions in cell culture and in the clinic.

 

During the last years, our emphasis has been on HCV given its magnitude as public health problem worldwide, and the direct implication of its quasispecies nature in the hepatic disease and response to treatments. The main contributions of our group to HCV have been: (i) that interferon resistance is multigenic; (ii) to make available HCV populations of different fitness level to study fitness effects on HCV biology; (iii) that viral fitness is a determinant of multi-drug resistance in absence of specific resistance mutations, (iv) the absence of population equilibrium (steady-state distribution of mutations and phenotypic stasis) even after extensive multiplication in a constant cellular environment, and (v) the design of sequential and combination treatments using both mutagenic and non-mutagenic antiviral agents for suppression of viral infectivity (summary in Figure 1). These investigations continue at the present time.

Lethal mutagenesis is an expanding field of research that has opened a new chapter in antiviral pharmacology. Our work, in collaboration with the late John Holland (UCSD), provided the first experimental evidence that an increase of mutation rate promoted by chemical mutagenesis was detrimental to RNA virus survival (1990-1997). In our current screening of mutagenic agents that have been licensed for human use, we have documented that the broad spectrum antiviral agent favipiravir (T-705) is mutagenic for FMDV and HCV, and can lead to the extinction of these viruses. We have collaborated with the group of Juan Carlos Sáiz (INIA) to show that favipiravir is also mutagenic for West Nile virus and can drive the virus towards extinction. We keep incorporating new, broad-spectrum, mutagenic and non-mutagenic agents for our antiviral designs, aimed at suppressing a broad range of RNA viral pathogens. A major unresolved issue is the integration of mutagenic and non-mutagenic inhibitors into antiviral designs to combat RNA viral diseases.

We have discovered a new mechanism of resistance to lethal mutagenesis of FMDV to ribavirin and 5-fluorouracil, consisting in selection of viral mutants that can modulate nucleotide incorporation to avoid the mutational bias produced by mutagenic nucleotides. This mechanism does not alter the amplitude of the mutant spectrum, and the corresponding adaptive flexibility. Interestingly, a joker mutation (meaning a given mutation that is repeatedly encountered when the virus is subjected to different selective constraints) in non-structural protein 2C modulated nucleotide incorporation in response to ribavirin mutagenesis, in absence of mutations in the viral polymerase. These observations emphasize the multiple adaptive resources that RNA viruses have to respond to hostile environments, and justify our ongoing efforts to search for antiviral designs to strongly suppress virus multiplication.

We have also continued collaboration with Susanna Manrubia (CNB, CSIC) that has represented a highly clarifying link between theoretical studies and experimental designs. In an interesting connection between virus expansion in the field and the clonal evolution of organisms, we have proposed for viruses a distinction between mechanistically active but inconsequential recombination, and evolutionary relevant recombination. Based on the available evidence, we think that mutation and recombination occur continuously during RNA virus replication, but that a majority of mutant and recombinant genomes do not survive (they are subjected to negative selection). However, there are subsets of mutations that combined with punctuated, biologically relevant recombination events, contribute to virus survival and evolution (Figure 2). These studies are contributing also to a deeper understanding of the molecular events underlying viral disease processes.

Our major aim is to use the new understanding of viral dynamics that has been provided by the framework of quasispecies theory to find new ways to suppress viral multiplication. It is our conviction that new antiviral designs, which are based on the understanding of the implications of quasispecies, can be used to control established, emergent, and re-emergent viral pathogens (Figure 3).

 

figura 1

Figure 1: Graphic summary of our recent work on quasispecies implications, and lethal mutagenesis of RNA viruses. The left panel illustrates HCV mutational waves (color-coded) quantified by molecular cloning-Sanger sequencing and deep sequencing during viral multiplication in a constant biological environment. The waves drawn here have been found by the two methods, excluding that they are due to a sampling bias. Panels on the right illustrate the conceptual basis of lethal mutagenesis, with an example of HCV extinction (top), a serial passage design (middle), and HCV extinction associated with ribavirin treatment (bottom). Detailed information is in the indicated references, and the reference list.

 

figura 2

Figure 2: Schematic representation of clonal evolution of viruses. From an initial infection (Origin, bottom) multiple sublineages are generated. Recombination takes place at any branch (double-headed arrows perpendicular to branches). Biologically relevant diversification is illustrated by red and blue branches. Recombination at a discontinuity point (large vertical double-headed arrow) is biologically relevant because it generates mosaic genomes with new phenotypes. Clonal evolution continues until a new discontinuity point is reached. The scheme does not imply a space or time scale. Reproduced from Perales et al. 2015, with permission from the National Academy of Sciences USA.

 

figura 3

Figure 3: A summary of the approach and main goal of our research.

 


 

Relevant publications (since 2012) grouped by topics 

Quasispecies implications / Implicaciones de cuasiespecies:

  • Domingo, E., Sheldon, J. and Perales, C. (2012). Viral quasispecies evolution. Microbiology and Molecular Biology Reviews, 76, (2)159-216.
  • Andino, R. and Domingo, E. (2015). Viral quasispecies. Virology, 479-480: 46-51.
  • Domingo, E. Perales, C. (2016). Viral quasispecies and lethal mutagenesis. European Review, 24(1):39-48.
  • Domingo, E. Perales, C. (2016). Species Concepts: Viral quasispecies. In: Kliman, R.M. (ed.), Encyclopedia of Evolutionary Biology, vol.4, pp. 228-235. Oxford: Academic Press.
  • Domingo, E. (2016). Virus as Populations. Composition, complexity, dynamics and biological implications. Academic Press, Elsevier, Amsterdam.
  • Domingo, E., Schuster, P. (eds.) (2016). Quasispecies: From Theory to Experimental Systems.
  • Domingo, E., Schuster, P. (2016). What is a quasispecies? Historical origins and current scope. Curr. Top. Microbiol. Immunol., 392:1-22.
  • Gregori, J., Perales, C., Rodríguez-Frías, F., Esteban, J.I., Quer, J., Domingo, E. (2016). Viral Quasispecies Complexity Measures. Virology 493: 227-237.
  • Domingo, E., de la Higuera, I., Moreno, E., de Ávila, A.I., Agudo, R., Arias, A., Perales, C. (2017). Quasispecies dynamics taught by natural and experimental evolution of foot-and-mouth disease virus. In: F. Sobrino and E. Domingo (Eds.). Foot-and-Mouth Disease Virus: Current Research and Emerging Trends, Horizon Scientific Press – Caister Academic Press, Poole, UK, pp. 147-170.
  • Moreno, E.; Gallego, I.; Gregori, J.; Lucia-Sanz, A.; Soria, M. E.; Castro, V.; Beach, N. M.; Manrubia, S.; Quer, J.; Esteban, J. I.; Rice, C. M.; Gomez, J.; Gastaminza, P.; Domingo, E.; Perales, C. (2017). Internal Disequilibria and Phenotypic Diversification during Replication of Hepatitis C Virus in a Noncoevolving Cellular Environment. J. Virol., 91 (10), e02505-16.
  • Domingo, E., Perales, C. (2018). Quasispecies and virus. Eur. Biophys. J. doi: 10.1007/s00249-018-1282-6.
  • Domingo, E., Perales, C. (2018). RNA virus genomes. In: Encyclopedia of Life Sciences. In press.
  • Domingo, E. and Perales, C. (2018). Virus evolution. Encyclopedia of Microbiology. 4th edition. In press.

Foot-and-mouth disease virus and the viral polymerase / Virus de la fiebre aftosa y polimerasa vírica:

  • Ferrer-Orta, C., de la Higuera, I., Caridi, F., Sanchez-Aparicio, M.T., Moreno, E. Perales, C., Singh, K., Sarafianos, S.G., Sobrino, F. Domingo, E., Verdaguer, N. (2015). Multifunctionality of a picornavirus polymerase domain: nuclear localization signal and nucleotide recognition. J. Virol., 89(13):6848-6859.
  • Sobrino, F. and Domingo, E. (eds.) (2017). “Foot-and-Mouth Disease Virus: Current Research and Emerging Trends”, Horizon Scientific Press – Caister Academic Press, Poole, UK.
  • De la Higuera, I., Ferrer-Orta, C., Moreno, E., De Avila, A.I., Soria, M.E., Singh, K., Caridi, F., Sobrino, F., Sarafianos, S.G., Perales, C., Verdaguer, N., and Domingo, E. (2018). Contribution of a multifunctional polymerase region of foot-and-mouth disease virus to lethal mutagenesis. J. Virol. 92(20). pii: e01119-18.

HCV virus-host interactions / Interacciones VHC-hospedador:

  • Madejón, A., Sheldon, J., Francisco-Recuero, I., Perales, C., Dominguez-Beato, M., Lasa, M., Sanchez-Perez, I., Muntané, J., Domingo, E., Garcia-Samaniego, J., Sanchez-Pacheco, A. (2015). Hepatitis C virus-mediated Aurora B kinase inhibition modulates inflammatory pathway and viral infectivity. Journal of Hepatology, 63(2): 312-9.
  • Perez-del-Pulgar, S., Gregori, J., Rodriguez-Frias, F., Gonzalez, P., García-Cehic, D., Ramirez, S., Casillas, R., Domingo, E., Esteban, J. I., Forns, X., Quer, J. (2015). Quasispecies dynamics in hepatitis C liver transplant recipients receiving grafts from hepatitis C virus infected donors. J. Gen. Virol., 96:3493-3498.
  • Valero, M.L., Sabariegos, R., Cimas, F., Perales, C., Domingo, E., Sánchez-Prieto, R., Mas, A. (2016). HCV RNA-dependent RNA polymerase interacts with Akt/PKB inducing its subcellular re-localization. 
  • Domínguez-Molina, B., Machmach, K., Perales, C., Tarancón-Díez, L., Gallego, I., Shedon, J., Leal M., Domingo E, and E. Ruiz-Mateos (2018). Toll Like Receptor-1 7 and -9 agonists improve hepatitis C virus replication and infectivity inhibition by plasmacytoid dendritic cells. J. Virol. pii: JVI.01219-18.

Drug resistance / Resistencia a drogas:

  • Perales, C., Beach, N. M., Gallego, I., Soria, M. E., Quer, J., Esteban, J. I., Rice, C., Domingo, E., and Sheldon, J. (2013). Response of hepatitis C virus to long-term passage in the presence of interferon-α. Multiple mutations and a common phenotype. J. Virol., 87(13), 7593-7607.
  • Sheldon, J., Beach, NM., Moreno, E., Gallego, I., Piñeiro, D., Martínez-Salas, E., Gregori, J., Quer, J., Esteban, JI., Rice, CM., Domingo, E., Perales, C. (2014). Increased replicative fitness can lead to decreased drug sensitivity of hepatitis C virus. J. Virol., 88(20):12098-12111.
  • Perales, C., Quer, J., Gregori, J., Esteban, J.I., Domingo, E. (2015). Resistance of hepatitis C virus to inhibitors: complexity and clinical implications. Viruses, 7:5746-5766.
  • Gallego, I., Sheldon, J., Moreno, E., Gregori, J., Quer, J., Esteban. J.I., Rice, C.M., Domingo, E., Perales, C. (2016). Barrier-Independent, Fitness-Associated Diferences in Sofosbuvir Efficacy against Hepatitis C virus. 
  • Martín, V., Perales, C., Fernández-Algar, M., Dos Santos, HG., Garrido, P., Pernas, M., Parro, V., Moreno, M., García-Pérez, J., Alcamí, J., Torán, JL., Abia, D., Domingo, E., Briones, C. (2016). An Efficient Microarray-Based Genotyping Platform for the Identification of Drug-Resistance Mutations in Majority and Minority Subpopulations of HIV-1 Quasispecies. PLoS ONE, 11(12):e0166902.
  • Perales, C., Chen, Q., Soria, M.E., Gregori, J., García-Cehic, D., Nieto-Aponte, L. Castells, L., Imaz, A., Llorens, M., Domingo, E., Buti, M., Esteban, J.I., Rodriguez-Frias, F., and J. Quer (2018). Baseline hepatitis C virus resistance-associated substitutions present at frequencies lower than 15% may be clinically significant. Infection and Drug Resistance. 11:2207-2210.
  • Perales, C. (2018) Quasispecies dynamics and clinical significance of HCV antiviral resistance. Int. J. Antimicrob. Agents. doi: 10.1016/j.ijantimicag.2018.10.005.
  • Soria, M.E., Gregori, J., Chen, Q., García-Cehic, D., Llorens, M., De Ávila, A.I., Beach, N.M., Domingo, E., Rodríguez-Frías, F., Buti, M., Esteban, R., Esteban, J.I., Quer, J., and C. Perales (2018). Pipeline for specific subtype amplification and drug resistance detection in hepatitis C virus. BMC Infectious Diseases. 18(1):446.
  • Perpiñán, E., Caro-Pérez, N., García-González, N., Gregori, J., González, P., Bartres, C., Soria, M.E., Perales, C., Lens, S., Mariño, Z., Londoño, M.C., Ariza, X., Koutsoudakis, G., Quer, J., González-Candelas, F., Forns, X., and S. Pérez-del-Pulgar.(2018). Hepatitis C virus early kinetics and resistance-associated substitution dynamics during antiviral therapy with direct-acting antivirals. J. Viral Hepatitis. doi: 10.1111/jvh.12986.

Lethal mutagenesis / Mutagénesis letal:

  • Ortega-Prieto, A.M., Sheldon, J., Grande-Pérez, A., Tejero, H., Gregori, J., Quer, J., Esteban, J.I., Domingo, E. and Perales, C. (2013). Extinction of hepatitis c virus by ribavirin in hepatoma cells involves lethal mutagenesis. PLoS ONE, 8(8): e71039.
  • Perales, C., Domingo, E. (2016). Antiviral strategies based on lethal mutagenesis and error threshold. Curr. Top. Microbiol. Immunol., 392:323-339.
  • Agudo, R., de la Higuera, I., Arias, A., Grande-Perez, A., Domingo, E. (2016). Involvement of a joker mutation in a polymerase-independent lethal mutagenesis escape mechanism. Virology 494:257-266.
  • De Ávila, A.I., Gallego, I., Soria, M.E., Gregori, J., Quer, J., Esteban, J.I., Rice, C.M., Domingo, E., Perales, C. (2016). Lethal mutagenesis of hepatitis C virus induced by favipiravir. PLoS ONE, 11 (10): e0164691.
  • de Ávila, A. I.; Moreno, E.; Perales, C.; Domingo, E. (2017). Favipiravir can evoke lethal mutagenesis and extinction of foot-and-mouth disease virus. Virus Res., 233, 105-112.
  • de la Higuera, I.; Ferrer-Orta, C.; de Avila, A. I.; Perales, C.; Sierra, M.; Singh, K.; Sarafianos, S. G.; Dehouck, Y.; Bastolla, U.; Verdaguer, N.; Domingo, E. (2017). Molecular and Functional Bases of Selection against a Mutation Bias in an RNA Virus. Genome Biol. Evol., 9 (5), 1212-1228.
  • Escribano-Romero, E., Jiménez de Oya, N., Domingo, E., Saiz, J.C. (2017). Extinction of West Nile virus by favipiravir through lethal mutagenesis. Antimicrob. Agents Chemother., 61(11). pii: e01400-17.
  • Gallego, I., Gregori, J., Soria, M.E., García-Crespo, C., García-Álvarez, M., Gómez-González, A., Valiergue, R., Gómez, J., Esteban, J.I., Quer, J., Domingo, E. and C. Perales (2018). Resistance of high fitness hepatitis C virus to lethal mutagenesis.Virology. 523:100-109.
  • Gregori, J., Soria, M.E., Gallego, I., Esteban, J.I., Quer, J., Perales, C., and E. Domingo (2018). Rare haplotype load as marker for lethal mutagenesis. PloS ONE. 13(10): e0204877.

General viral evolution / Evolución general de virus:

  • Perales, C. Moreno, E. Domingo, E. (2015). Clonality and intracellular polyploidy in virus evolution and pathogenesis. Proc Natl Acad Sci USA, 112(29):8887-92.

 

 


 

Patents:

  • N. Sevilla, E. Domingo, C. Escarmís, S. Ojosnegros, J. García-Arriaza, M. Sanz-Rojo, T. Rodríguez. "Vacuna atenuada para la fiebre aftosa". Nº DE SOLICITUD: P200801583. Patente concedida en España el 16/06/2011. Nº PUB: ES2344875.

 


 

Doctoral Theses:

  • Héctor Moreno Borrego (2012).  Dinámica poblacional del virus de la coriomeningitis linfocitaria del ratón en su interacción con agentes mutagénicos. Universidad Autónoma de Madrid. Directors: Esteban Domingo y Verónica Martín.
  • Ignacio de la Higuera Hernández (2014). Factores determinantes del reconocimiento de nucleótidos en el virus de la fiebre aftosa. Universidad Autónoma de Madrid. Director: Esteban Domingo.
  • Ana Mª Ortega Prieto (2014). Mutagénesis letal del virus de la Hepatitis C. Universidad Autónoma de Madrid. Directors: Esteban Domingo y Celia Perales.
  • Elena Moreno del Olmo (2017). Evolución a largo plazo de virus RNA en ambiente biológico constante. Universidad Autónoma de Madrid. Directors: Esteban Domingo y Celia Perales.

 


 

Other Activities:

  • Académico Numerario de la Real Academia de Ciencias Exactas, Físicas y Naturales, adscrito a la Sección de Ciencias Naturales, (2011).
  • Miembro de la Red Española de Biofísica, coordinada por el Dr. David Reguera, desde 2011.
  • Miembro del Global Virology Network, coordinado por el Dr. Robert Gallo, desde 2011.
  • Miembro del Comité Organizador del Congreso FEMS 2011 (Ginebra, Suiza, 2011).
  • Editor asociado de la revista Virus Research desde 2012.

Biotechnology and genetics of extreme thermophiles

 grupo400

 


José Berenguer Carlos

BSciStaff

BPublications

 

Scientific abstract:

The main aim of our group is focused on the use of thermophiles as biological models and as tools and source of thermozymes for biotechnology. Our laboratory model is Thermus thermophilus (Tth) a thermophilic bacterium of ancestral phylogenetic origin that shows fast growth under laboratory conditions, and highly efficient natural competence apparatus (NCA).

 

figure 1

Figure 1: Extracellular DNA that enters Tth by the NCA faces its degradation by ThAgo through gDNA-DNA interference. In contrast, dsDNA transferred by a donor Tth cell through the TdtA translocase and incorporated through the NCA is not susceptible to ThAgo. The DNA helicase HepA is also required in the donor, likely to repair the scars generated in the genome in the almost random donation process.

 

As biological models, we have focussed our attention on a mechanism of horizontal gene transfer (HGT) between Tth strains that requires cell to cell contacts and depends on an active NCA in the recipient cell. The mechanism, named transjugation, also needs the presence in the donor cell of the a DNA translocase (TdtA) encoded along a restriction system and a nuclease in an operon belonging to a small Conjugative and integrative Element (ICEth1), which mechanism of transference is currently under study. Another point of attention in this basic biology studies is the role as barrier against HGT played by a homologue to human Argonaute (ThAgo). We know that ThAgo inhibits the entry of extracellular DNA through the NCA by a DNA-DNA interference mechanism. As this barrier action of ThAgo is bypassed when the DNA is transferred by transjugation, a mechanism of activation/deactivation has been hypothesized which is also the subject of our current research. In this context, TthAgo seems to distinguish between potentially dangerous environmental DNA from “trustworthy” DNA obtained from a related strain. Also, as nucleic acid interference constitutes the bases for gene edition, we are working on the use of this and other prokaryotic Ago for this purpose.

 

figure 2

Figure 2: The use of microdroplets in high-throughput screenings. A) Growth of microorganisms and mammalian cells. B) Screening for recombinant thermozymes expressed in Tth. C) In vitro expression of thermozymes using in vitro transcription/translation derived from Tth. D) Digital PCR to detect rare microorganisms (dark biological matter) and genes. Figure credits: M. Almendros, A. Lopes and M. Sánchez.

 

A major effort in our group is focused in the discovery of thermostable enzymes (thermozymes) and thermostable variants of enzymes that could better respond to the requirements for industrial biocatalysis. For this, we have used Tth as host for the in vivo selection of thermostable enzymes in large libraries of man-made diversity using newly-developed tools and strains. In parallel, we have incorporated to our laboratory methods of ultrahigh- throughput enzyme screening based on the use of water-in-oil emulsions or microdroplets. The reduction of the test tube to a compartment of picoliters enables performing up to 106 enzyme assays per day and paves the way for future single-cell biology experiments. Microdroplets are generated using self-made silicone microfluidic devices and screened on-chip using a bespoke FACS-like sorter.

 


 

Selected articles from the last 4 years:

  • Baquedano I, Mencía M, Blesa A, Burrus V and Berenguer J (2019) ICETh1 & ICETh2, two interdependent mobile genetic elements in Thermus thermophilus transjugation. Environ Microbiol doi: 10.1111/1462-2920.14833
  • Mate DM, Rivera NR, Sanchez-Freire E, Ayala JA, Berenguer J and Hidalgo A (2019) Thermostability enhancement of thePseudomonas fluorescensesterase I by in vivo folding selection inThermus thermophilus. Biotech. Bioeng. doi: 10.1002/bit.27170
  • Verdú C, Sanchez-Freire E, Ortega C, Hidalgo A, Berenguer J* and Mencía M* (2019) A modular vector toolkit with a tailored set of thermosensors to regulate gene expression in Thermus thermophilus. ACS Omegadoi: 10.1021/acsomega.9b02107
  • Alvarez L, Sanchez-Hevia D, Sánchez M and Berenguer J (2018) A new family of nitrate/nitrite transporters involved in denitrification. Int. Microbiol. 22, 19–28. doi: 10.1007/s10123-018-0023-0
  • Chahlafi Z, Alvarez L, Cava F and Berenguer J (2018) The role of conserved proteins DrpA and DrpB in nitrate respiration of Thermus thermophilus. Environ. Microbiol. 20, 3851- 3861. doi: 10.1111/1462-2920.14400
  • Antonucci I, Gallo G, Limauro D, Contursi P, Ribeiro AL, Blesa A, Berenguer J, Bartolucci S, and Fiorentino G (2018) Characterization of a promiscuous cadmium and arsenic resistance mechanisms in Thermus thermophilus HB27 and potential application of a novel bioreporter system. Microb. Cell Fact. 17, 78- 87. doi: 10.1186/s12934-018-0918-7
  • Blesa A, Averhoff B and Berenguer J (2018) Horizontal gene transfer in Thermus sp. Curr. Issues Mol. Biol. 29, 23-36. doi: 10.21775/cimb.029.023
  • Consolati T, Bolívar JM, Petrasek Z, Berenguer J, Hidalgo A, Guisán JM and Nidetzky B (2018) Bio-based pH internally sensitive materials: immobilized yellow fluorescent protein as optical sensor for spatiotemporal mapping of pH inside porous matrices. ACS Appl. Mater. Interfaces 10, 6858-6868. doi:10.1021/acsami.7b16639
  • Antonucci I, Gallo G, Limauro D, Contursi P, Ribeiro AL, Blesa A, Berenguer J, Bartolucci S and Fiorentino G (2017) An ArsR/SmtB family member regulates arsenic resistance genes unusually arranged in Thermus thermophilus HB27. Microb. Biotechnol. 10, 1690-1701. doi: 10.1111/1751-7915.12761
  • Alvarez L, García-Quintáns N, Blesa A, Baquedano I, Bricio C, Mencía M and Berenguer J (2017) Hierarchical control of nitrite respiration by transcription factors encoded within mobile gene clusters of Thermus thermophilus. Genes 8, 361. doi:10.3390/genes812036
  • Ribeiro AL, Sánchez M, Hidalgo A and Berenguer J (2017) Stabilization of enzymes by using thermophiles. Methods Mol. Biol. 1465, 297-312. doi: 10.1007/978-1-4939-7183-1_21
  • Blesa A, Quintáns NG, Baquedano I, Mata CP, Castón JR and Berenguer J (2017) Role of archaeal HerA protein in the biology of the bacterium Thermus thermophilus. Genes 8, 130. doi:10.3390/genes8050130
  • Blesa A, Baquedano I, García-Quintáns N, Mata CP, Castón JR and Berenguer J (2017) The transjugation machinery of Thermus thermophilus: Identification of TdtA, an ATPase involved in DNA donation. PloS Genetics 13, e1006669. doi: 10.1371/journal.pgen.1006669
  • Berenguer J, Mencía M and Hidalgo A (2017) Are in vivo selections on the path to extinction? Microb. Biotechnol. 10, 46-49. doi: 10.1111/1751-7915.1249

 


 

Last Doctoral Theses:

  • Alba Blesa Esteban (2016) Horizontal gene transfer in Thermus thermophilus: mechanisms and barriers. Universidad Autónoma de Madrid. Director: José Berenguer.
  • Yamal Al-ramahi González (2013) Engineering of fluorescent proteins and applications in cellular localization studies in thermophilic microorganisms. Universidad Autónoma de Madrid. Directors: José Berenguer and Aurelio Hidalgo.
  • Noé R. Rivera (2013) Thermal stabilization of proteins with biotechnological interest. Universidad Autónoma de Madrid. Directors: José Berenguer and Aurelio Hidalgo.
  • Laura Alvárez Múñoz (2012) Analysis of nitrite respiration in Thermus thermophilus. Universidad Autónoma de Madrid. Director: José Berenguer.

 


 

International Projects and Excellence networks:

  • METAFLUIDICS: Advanced toolbox for rapid and cost-effective functional metagenomic screening: microbiology meets microfluidics. Funding: European Union H2020. GA 685474. Period: 01/06/2016 to 30/05/2020. Coordinator: A. Hidalgo.
  • CARBAZYMES: Sustainable industrial processes based on a C-C bond-forming enzyme platform. Funding: European Union H2020. GA 635595. Period: 01/04/2015 to 31/03/2019. Role: Partner.
  • RedEx: National Network on Extremophilic Microorganisms. National project on excellence networks. BIO2015-71815. Period: 01/01/2016 to 30/06/2018. Role: Partner.
  • HOTDROPS: Ultrahigh-throughput platform for the screening of thermostable proteins by thermophilic in vitro transcription-translation and microfluidics. Funding: European Union FP7. GA 324439. Period: 01/06/2013 to 30/05/2017. Coordinator: J. Berenguer.

Molecular ecology of extreme environments

 

grupo-400

 


Ricardo Amils

BSciStaff

BPublications

 

Research summary:

Molecular Ecology of Extreme Environments: This area of research has the following objectives:
- Acidophiles: conventional microbial ecology, molecular ecology, molecular biology and biotechnology (bioleaching, specific metal sequestering and phytoremediation) of extreme acidic environments (Río Tinto basin, different mine sites of the Iberian Pyritic Belt, río Agrio (Argentina), Antartica),

fig01-300  -------  fig02-300
Obtención de muestras del subsuelo en condiciones anaerobias.   CARD-FISH en una muestra de -284m de profundidad.


- Geomicrobiological characterization of extreme environments as habitability models: Tinto basin (Mars analogue), sulfide deposits from Antartica (Mars analogue), Tirez hypersaline lagoon and Uyuni salt lake (Europa analogues), both in collaboration with professor I. Marín (UAM), permafrost areas of Alaska (Mars analogue).
- Geomicrobiology of the Iberian Pyritic Belt (IPB) subsurface: characterization of the subsurface bioreactor responsible of the extreme acidic conditions of Río Tinto. This work is done in collaboration with the Centro de Astrobiología (ERC Project IPBSL)
- The line of microbial ecology of anaerobic environments directed by professor J.L. Sanz (UAM) is being developed in the 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, subsurface of the IPB).
Micology, This area of research directed by Dr. Aldo González 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.
- Transcriptomics and proteomics for the study of the secretome


 Selected Publications:

  • Sánchez-Román, M., Romanek, C.S., Fernández-Remolar, D., Sánchez-Navas, A., McKenzie, J.A., Amils, R., and Vasconcelos, C. (2011) Chemical Geology, 281 :143-150. doi:10.1016/j.chemgeo.2010.11.020.
  • Fernández-Remolar, D., Prieto-Ballesteros, O., Gómez-Ortiz, D., Fernández-Sampedro, M., Sarrazin, P., Gailhanou, M., and Amils, R. (2011) Icarus, 211: 114-138.
  • González-Toril, E., Aguilera, A., Souza-Egipsy, V., López Pamo, E., Sánchez-Espada, J., and Amils, R. (2011) Appl. Environ. Microbiol., 77: 2685-2694.
  • Souza-Egipsy, V., Altamirano, M., Amils, R., and Aguilera, A. (2011) Environ. Microbiol., 13(8): 2351-2358, doi:10.1111/j.1462-2920.2011.02506.x.
  • García-Muñoz, J., Amils, R., Fernández, V.M., De Lacey, A., and Malki, M. (2011) Internat. Microbiol., 14: 73-81.

 Doctoral Theses:

Irene Sánchez-Andrea (2012). Diversidad microbiana de los sedimentos anaerobios del Río Tinto. Universidad Autónoma de Madrid, José Luis Sanz y Ricardo Amils.


 Other activities:

Editor de Encyclopedia of Astrobiology, Springer, 2011.

Viral modulation of the immune response

 

 grupo-400

 


 

 

Antonio Alcamí

BSciStaff

Blistado

 

 

 

 

Research summary:

We are investigating immune evasion mechanisms employed by large DNA viruses, poxviruses and herpesviruses. Specifically, we are characterizing viral proteins that are secreted from infected cells, interact with cytokines and chemokines, and control their immunomodulatory activity. We work on two virus systems: (1) Herpesviruses like herpes simplex virus, a human pathogen of clinical relevance; and (2) Poxviruses such as vaccinia virus, the smallpox vaccine. These viral cytokine receptors have unexpected properties, enhancing the activity of chemokines or binding to the cell surface to be retained in the vicinity of infected tissues, and provide insights into the function of cytokines. The contribution of viral cytokine receptors to pathogenesis and immune modulation is being addressed in mice infected with ectromelia virus, a natural mouse pathogen that causes a smallpox-like disease known as mousepox.

fig01-300  
Virus-encoded chemokine binding proteins.  


Viruses offer a unique opportunity to develop their immune evasion strategies, optimized for millions of years of evolution, as novel therapeutic approaches. In collaboration with Biotech Companies, we are developing viral immunomodulatory proteins as potential medicaments to treat human allergic and autoimmune diseases.
We are sequencing the complete genome of large DNA viruses in order to identify new viral genes involved in pathogenesis and immune modulation, including natural isolates of ectromelia virus and new iridoviruses infecting fish and amphibian. Viruses are the most abundant and diverse biological entities on Earth. Following metagenomic approaches, we are characterizing complex viral communities using next generation sequencing methodologies (454-Roche, Illumina). We 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. Viral metagenomics is being used to identify viruses associated with human pathologies, such as multiple sclerosis.

 

 -- fig02-300
 

Electron micrograph of an ectromelia virus-infected cell showing inclusion body with mature virus particles.

 

 

 

 

 


 Selected Publications:

  • Alcami, A. and Moss, B. (2011) Smallpox Vaccines. In: Khan, A. S. and Smith, G. L. (eds) Scientific Review of Variola Virus Research 1999-2010. World Health Organization, Geneva, Switzerland, pp. 1-15.
  • Alejo, A., Pontejo, S. M., and Alcami, A. (2011) Poxviral TNFRs: properties and role in viral pathogenesis. Adv. Exp. Med. Biol.691, 203-210.
  • Montanuy, I., Alejo, A., and Alcami, A. (2011) Glycosaminoglycans mediate retention of the poxvirus type I interferon binding protein at the cell surface to locally block interferon antiviral responses. FASEB J. 25, 1960-1971.
  • Xu, R., Rubio, D., Roscoe, F., Krouse, T. E., Truckenmiller, M. E., Norbury, C. C., Hudson, P. N., Damon, I. K., Alcami A., and Sigal, L. J. (2012) Antibody inhibition of a viral type I interferon decoy receptor cures a viral disease by restoring interferon signaling in the liver. PLoS Pathogens 8(1):e1002475.
  • Viejo-Borbolla, A., Martinez-Martín, N., Nel, H. J., Rueda, P., Martín, R., Blanco, S., Arenzana-Seisdedos, F., Thelen, M., Fallon, P. G., and Alcami, A. (2012) Enhancement of chemokine function as an immunomodulatory strategy employed by human herpesviruses. PLoS Pathogens 8(2): e1002497.

 


 

Patents:

- Martín-Pontejo, S. y Alcami, A. Unión a glicosaminoglicanos de proteínas con dominio SECRET codificadas por poxvirus. Número de prioridad: P201230540. País: España. Fechas de prioridad: 11 abril 2012. Propietario: CSIC.
- Cabrera, J. R., Viejo-Borbolla, A., Martínez-Martín, N., Wandosell, F. y Alcami, A.. 'Proteína viral recombinante SgG2 y/o complejos binarios SgG2-FNs para su uso en crecimiento y/o regeneración axonal'. Número de p rioridad: P201231654. País: España. Fecha de prioridad: 26 octubre 2012. Propietario: CSIC.


 

Doctoral Theses:

Sergio Martín Pontejo (2012). Características moleculares y funcionales de los receptores solubles del TNF con capacidad anti-quimioquinas de poxvirus. Universidad Autónoma de Madrid. Directores: Begoña Ruiz Argüello y Antonio Alcamí.

Marcos Palomo (2012). Caracterización de inhibidores solubles de interferón codificados por poxvirus. Universidad Autónoma de Madrid. Director: Antonio Alcamí.

Nadia Martínez Martín (2012). Herpes simples virus glycoprotein G enhances chemotaxis and axonal growth through modification of plasma membrane microdomains and receptor trafficking. Universidad Autónoma de Madrid. Directores: Abel Viejo Borbolla y Antonio Alcamí.


 

Other activities:

- Member of the Editorial Board of Virology
- Member of the Editorial Board of Journal of Virology
- Advisor to the World Health Organization Advisory Committee on Variola Virus Research
- Organizer, together with R. Blasco and E. Villar, of the XIX International Poxvirus, Asfarvirus and Iridovirus Conference. Salamanca, June 2012.
- The Group participates in the Spanish Network of Multiple Sclerosis (www.reem.es)

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