Caracterizacion de la proteina 3a del virus de la fiebre aftosa. Estudio de su dimerizacion, capacidad de unio a membranas y dinamica celular

• Autores: Mónica González Magaldi
• Directores de la Tesis: Francisco Sobrino Castelló (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2012
• Idioma: español
• Tribunal Calificador de la Tesis: Isabel Correas Hornero (presid.), José Antonio López Guerrero (secret.), Alejandro Brun Torres (voc.), Cristina Risco Ortiz (voc.), Núria Verdaguer Massana (voc.)
• Materias:
  • Ciencias de la vida
    • Virología
• Enlaces
  • Tesis en acceso abierto en: digitool-uam.greendata.es
• Resumen

  • Foot-and-mouth disease virus (FMDV) is a positive single strand RNA virus that belongs to the Picornaviridae family and causes a highly transmissible and damaging livestock disease. Its genome codes for structural and non structural (NSP) viral proteins. NSP 3A can play different roles in picornavirus replication and growth, including interactions with cell membranes through its hydrophobic region. FMDV 3A is unique among picornaviruses in that its C-terminal region is considerably longer and it is linked to three tandem-copies of 3B, instead of a single one. Homodimerization of 3A has been shown to be relevant for its biological activity in enteroviruses such as poliovirus and coxsackie virus B3.

    In this study, we have attempted to gain insight into the poorly understood structure- function relationship of FMDV 3A. Structural analyses from FMDV 3A expressed in E.coli were impaired by the difficulties found for its purification. Nevertheless, these experiments provided the first evidence on the 3A capacity to form dimers. In addition, the analysis of the effect of the expression of 3A and fragments of this protein revealed that its N-terminal fragment induced membrane alterations in bacterial cells.

    Homodimerization of FMDV 3A in susceptible cultured cells was evidenced by an in situ protein fluorescent ligation assay in infected cells, as well as in cells transiently expressing this protein. A molecular model of the N-terminal fragment of FMDV 3A protein, derived from the structure of the poliovirus 3A protein, predicted a hydrophobic interface spanning residues 25 to 44 as the main determinant for 3A dimerization. Replacements L38E and L41E, involving charge acquisition at residues predicted to contribute to the hydrophobic interface, reduced the dimerization signal in the protein ligation assay, and prevented the detection of dimer/multimers species in both transiently expressed 3A proteins and in synthetic peptides reproducing the N-terminus of 3A.

    On the other hand, transiently expressed FMDV 3A behaved in a polyvalent manner in experiments aimed to study its interactions with cell membranes. Thus, only about 50% of the protein was recovered in the soluble fraction of transfected cells and treatment of the insoluble protein with different agents rendered two fractions with different solubilization patterns, each corresponding to peripheral and integral membrane proteins, respectively.

    Finally, biophysical studies of protein dynamics in living cells using fusions with GFP revealed that 3A showed a confined movement, with a mobile fraction determined by FRAP analyses of about 40%. In addition, 3A exhibited characteristics of a protein mainly associated to a continuous compartment in the cell, as reveled by FLIP analyses.