Análisis de los factores y dominios implicados en la interacción homomérica de la arn-polimerasa del virus de la hepatitis c

• Autores: Itxaso Bellon Echeverria
• Directores de la Tesis: Antonio Mas López (dir. tes.)
• Lectura: En la Universidad de Castilla-La Mancha ( España ) en 2011
• Idioma: español
• Tribunal Calificador de la Tesis: Esteban Domingo Solans (presid.), Victoriano Baladrón García (secret.), María del Rosario Sabariegos Jareño (voc.), Eva María Galán Moya (voc.), Ricardo Sánchez Prieto (voc.)
• Materias:
  • Química
    • Bioquímica
      • Proteínas
  • Ciencias de la vida
    • Biología molecular
    • Virología
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• Resumen

  • Hepatitis C virus is a positive strand RNA virus that replicates its genome in replication complexes associated to endoplasmic reticulum-derived micro-vesicles. A key protein in these complexes is NS5B, the viral RNA-dependent RNA-polymerase. Recently, it has been reported that NS5B interacts with itself forming oligomers. Mutations that disrupt these interactions were lethal for polymerase function. Therefore, NS5B oligomerization could be a new target for the design of anti-HCV compounds.

    In this Thesis, we have developed a new accurate and quantitative method to measure NS5B-NS5B interactions by using Förster-resonance-energy-transfer (FRET) in vitro using recombinant proteins. This method has allowed us to analyse the conditions driving the interactions between NS5B polymerases. The most important interactions among monomers are electrostatic forces. Both NaCl and KCl led to concentration-dependent changes in the oligomerization status of NS5B. We have also tested different combinations of point mutants showing FRET values from zero to around 100%. Furthermore, cooperativity in RNA synthesis activity has been analysed by determining the Hill coefficient and the results were consistent with those obtained for oligomerization. We have extended these studies to HCV RNA-polymerases from different genotypes (genotypes 1 to 5). Finally, we also analyse the effect on NS5B oligomerization of reaction conditions needed for de novo and primer extension RNA-polymerase activity, as well as in the presence of non-nucleoside inhibitors.

    In summary, we have developed a method that allows us to measure the effect of different conditions and/or compounds on NS5B oligomerization. We have related oligomeric structures to de novo initiation conditions. Moreover, we have described that some inhibitors target NS5B oligomerization. Finally, results obtained from NS5B point mutants together with data reported by other groups allowed us to infer the geometry of the NS5B homomeric interaction.