Structural and functional analysis of Zika virus NS5 protein

• Autores: Victor Manuel Ruiz Arroyo
• Directores de la Tesis: Núria Verdaguer Massana (dir. tes.), Diego Sebastián Ferrero (codir. tes.)
• Lectura: En la Universitat de Barcelona ( España ) en 2020
• Idioma: inglés
• Tribunal Calificador de la Tesis: Ignacio Fita Rodríguez (presid.), Antonio Mas López (secret.), Alba Guarne Cabello (voc.)
• Programa de doctorado: Programa de Doctorado en Biotecnología por la Universidad de Barcelona
• Materias:
  • Química
    • Química analítica
      • Espectroscopia de resonancia magnética
      • Microscopia
    • Bioquímica
      • Proteínas
  • Ciencias de la vida
    • Virología
      • Arbovirus
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• Resumen

  • Zika virus (ZIKV) belongs to the Flaviviridae family and constitute an important public health concern since ZIKV infection produced devastating effects in new born infants. Flaviviruses present a positive sense single stranded RNA genome flanked by highly structured untranslated regions (UTR) carrying one open reading frame that codifies for three structural proteins (C, prM, E) and five nonstructural proteins (NS1-5). At the most C-terminal end, NS5 protein carries a RNA dependent RNA polymerase (RdRP) and a methyl transferase domain (MTase) for genome copying and 5’ capping activities of the newly synthesized RNA, respectively. Given the crucial role of this enzyme for viral replication, NS5 constitutes an attractive antiviral target to inhibit viral replication. In this study, we determined the structure of the ZIKV NS5 protein using X-Ray crystallography combined with several structural biology approaches to characterize the supramolecular arrangement of the ZIKV NS5 protein. We identified the monomer-monomer and dimer-diner interactions to form fibril-like structures, and evaluated the role of oligomer formation, using in-vitro polymerization assays. We also evaluated the in-vivo effect of NS5-oligomerisation in chicken embryos, stablishing a connection between this protein and microcephaly.

    One of the most important RNA structures present at the 5’UTR of flavivirus genomes is the 5SLA. This structure was identified previously to bind the NS5 protein, acting as a promoter and being essential for viral replication. We assayed and optimized the NS5-5SLA complex stability using biophysical and biochemical techniques and determined the structure of the complex by single particle cryo- EM. Comparisons between the NS5-5SLA complex and the NS5 crystallographic structure revealed for the first time in flavivirus, important conformational changes in the NS5 RdRP. We identified the residues involved in complex formation and characterized the effect of this binding on NS5 polymerization, shedding new light on the understanding of replication mechanisms in flaviviruses.