Análisis del motivo de unión al dsRNA de la proteína VP3 del virus de la Bursitis Infecciosa y su implicación en el control de la respuesta innata antiviral del hospedador

• Autores: Idoia Busnadiego Rezola
• Directores de la Tesis: José Francisco Rodríguez Aguirre (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2013
• Idioma: español
• Tribunal Calificador de la Tesis: Núria Verdaguer Massana (presid.), Susana Guerra García (secret.), Daniel Luque Buzo (voc.), Fernando Almazán Toral (voc.), Natàlia Majó i Masferrer (voc.)
• Materias:
  • Ciencias de la vida
    • Biología molecular
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • ABSTRACT/RESUMEN EN INGLÉS Infectious bursal disease virus (IBDV) is an avian pathogen responsible for an acute immunosuppressive disease that causes major losses to the poultry industry. IBDV belongs to the Birnaviridae family, which includes nonenveloped viruses possessing a bisegmented double-stranded RNA (dsRNA) genome enclosed within a single icosahedral T = 13 capsid lacking the characteristic T = 2 core present in all known icosahedral dsRNA viruses that isolates the virus genome and replicative intermediates from host sensors. The inner space of birnavirus virions is occupied by ribonucleoprotein (RNP) complexes formed by the genomic dsRNA segments entirely wrapped up by molecules of the virus encoded VP3 polypeptide and covalently linked to the VPg form of the viral RNA-dependent RNA-polymerase. Previous reports have demonstrated that these RNPs are transcriptionally active in vitro and in vivo in the absence of an intact capsid and this suggests the possibility that the RNPs might be released and act as capsid-independent transcriptional complexes. In this regard, we raised the hypothesis that the VP3 polypeptide might play a role shielding the dsRNA against cellular dsRNA sensors, and thus preventing the onset of dsRNA-mediated innate immune responses.

    Previous reports evidenced that expression of the mature VP2 IBDV capsid polypeptide triggers a programmed cell death response. Our results demonstrate that coexpression of the VP3 polypeptide precludes phosphorylation of both PKR and eIF2¿ induced by VP2 expression. Further experiments showed that VP3 functionally replaces the host-range vaccinia virus (VACV) E3 protein, thus allowing the E3 deficient VACV deletion mutant WR/¿E3L to grow in non-permisive cell lines. We also show that when analyzed in a plant-based experimental model, VP3 prevents the silencing-mediated degradation of a reporter mRNA and can functionally replace the well characterized HCPro silencing suppressor of Plum pox virus, a potyvirus that is unable to infect plants in the absence of an active silencing suppressor. We also demonstrate that the anti-apoptotic and anti-silencing activities of VP3 rely on its dsRNA binding capacity. Finally, through the employment of surface plasmon resonance (SPR) analysis, we obtained the kinetic constants of the VP3-dsRNA interaction and determined that K99 and K106 are the key VP3 residues for binding the dsRNA.

    According to results presented here, VP3 can be categorized along with other well characterized proteins such as VACV E3, avian retrovirus sigmaA, and influenza virus NS1 as a virus-encoded dsRNA-binding polypeptide with antiapoptotic properties. Our results suggest that VP3 plays a central role in ensuring the viability of the IBDV replication cycle by preventing programmed cell death protecting the viral genome from host sentinels.