Contribuciones al estudio de la funcionalidad de proteínas no estructurales del virus de la fiebre aftosa

• Autores: Yuri Allende Vieira Prado
• Directores de la Tesis: María Flora Rosas Kuz (dir. tes.), Francisco Sobrino Castelló (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2015
• Idioma: español
• Tribunal Calificador de la Tesis: José María Almendral del Río (presid.), José Ignacio Núñez Garrote (secret.), Alejandro Brun Torres (voc.), Esther Blanco Lavilla (voc.), Ángel Luis López Carrascosa (voc.)
• Materias:
  • Ciencias agrarias
    • Ciencias veterinarias
      • Virología
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Foot-and-mouth disease virus (FMDV) is a positive strand RNA virus that belongs to the picornaviridae family. The FMDV RNA encodes structural and nonstructural proteins (NSP). In picornaviruses, the NSP 3A contributes to the cell rearrangements associated to the infection and is responsible for anchoring the replication complex to intracellular membranes. This protein contains a hydrophobic domain and shows the ability to form antiparallel dimers, whose function is not yet known. To study the functional role of residues involved in this dimerization, mutations were introduced in the infectious clone available in the laboratory (plasmid pMT28) at residues M29 and I42 of 3A, that are located at the hydrophobic ¿-helices predicted to contribute to dimer stability. Replacements M29D and I42D that introduce negative charges were lethal to FMDV. The mutations to positive charges, M29R and I42R, were viable in only one of three independent assays performed. The viruses recovered from mutant I42R showed the reversion to a hydrophobic residue (R42L), while that recovered from mutant M29R selected two additional mutations: replacement I42L, in a residue predicted to interact with M29, and S140F. These results support the relevance of the hydrophobic interface formed by residues 25-33 and 37-44 of 3A for FMDV multiplication.

    Previous results indicated that introduction of a Gly at residue 5 within the sequence ISIP - predicted as target cleavage 2C/3A by viral protease 3C - was lethal for viral RNA infectivity. Here, we have determined that the analogous insertion of a Gly at 3A residue 151, corresponding to the equivalent sequence (PQAE) predicted for cleavage 3A/3B1 (mutant 3A-¿G) rendered RNAs infectious in cultured cells. Upon serial passages in BHK-21, 3A-¿G mutant exhibited a kinetic growth similar to that of the wt virus, despite showing an abolished 3A/3B1 processing, indicating that efficient virus multiplication can occur in the presence of small amounts of mature 3A protein.

    Transient expression of viral proteins is an approach widely used to study the requirements for viral replication. To explore the feasibility of reconstructing a cellular environment in which different FMDV nonstructural proteins were transiently expressed, we attempted the cloning and expression in pRSV of 3ABC and P3 (3ABCD) FMDV protein precursors. All the pRSV inserts either incorporating 3ABC or P3, found in the positive bacterial clones showed a variety of mutations in the viral 3C protease. The only single aminoacid replacements were found at residues - L151F, H181Y and S182F - involved in the protease activity of the protein, suggesting that the toxicity exerted by 3C drove the selection of mutations that decreased such effect. Indeed, replacements L151F, H181Y and S182F severely decreased 3C protease activity while they were lethal for RNA infectivity in cultured cells.

    Finally, the use of viral RNA replicons as an alternative tool for the study of the interactions between FMDV and the host cell was explored. Thus, replicons in which the capsid protein coding region (P1) was replaced by the CAT gene sequence and that included or not NSP L, were constructed from plasmid pMT28. The preliminary results presented indicate that the replicon lacking the L gene produced higher levels of CAT and NSP than that including this protein.