Estudio de proteínas no estructurales del virus de la fiebre aftosa|banálisis funcionales y aplicación al diagnóstico viral

Abstract

Foot-and-mouth disease virus (FMDV) is a positive strand RNA virus belonging to the family Picornaviridae. Despite picornavirus multiplication entirely occurs in the cytoplasm of infected cells, recent evidences indicate that these viruses can interact with the nuclei of host cells, altering the traffic nucleus/cytoplasmic and reprogramming the cell nucleus, i.e. to inhibit cellular immune responses. We previously reported that FMDV 3D protein, the viral RNA polymerase RNA dependent, can be detected in the nucleus of infected cells. In this Doctoral Thesis we have experimentally tested whether the MRKTKLAPT sequence in 3D (residues 16 to 24), predicted as a potential nuclear localization signal (NLS) by its homology with related signals recently reported for other picornaviruses, could actually act as a NLS. To this end, mutations leading to substitutions in the two amino acid residues within this sequence, K18E and K20E, were introduced in the full length infectious clone pMT28. Each of these substitutions resulted detrimental for viral RNA replication and virus production in BHK-21 cells, and led to the recovery of viruses with a direct reversion of the mutation. When 3D substitutions K18E and K20E were introduced in plasmid pRSV a decreased nuclear localization was observed in the transient expression of 3D and its precursor 3CD, suggesting the role of residues K18 and K20 in nuclear targeting. Finally, fusion of MRKTKLAPT to the green fluorescence protein (GFP) modified the cellular distribution of GFP increasing its nuclear location, which was not observed when GFP was fused to the corresponding 3D mutated sequences. These results indicate that the sequence MRKTKLAPT can be functionally considered as a NLS. The effect of FMDV infection on the traffic nucleus/cytoplasm and on proteins forming the nuclear pore complex (NPC) was also studied. A disruption was observed in the classical nuclear import pathway indicated by the decrease in the nuclear localization of a recombinant GFP fused to the SV40 NLS. In addition, the levels of expression and the subcellular location of the nucleoporin Nup153 were found altered in infected cells. Detection of infection-specific FMDV antibodies is a key issue for the control of FMD, which causes one of the most damaging and feared animal diseases. Antibodies to FMDV NS proteins, mainly those to the 3AB region, have been shown to permit specific serological distinction between infected and conventionally vaccinated animals, by conventional techniques. Biosensors constitute a promising and alternative tool as they can allow development of fast and species-independent diagnostic tests. The second part of this Doctoral Thesis describes the analysis of recombinant ßgalactosidases accommodating one or two different peptide sequences from FMDV 3B protein at two independent insertion sites in the enzyme. These enzymes showed a decreased activity that correlated with higher-lever reactivations in the presence of monoclonal antibodies or sera from different natural FMDV host species. We have studied enzymatic reaction conditions to found the optimal ones to optimize the enzymatic activity, achieving greater differences between the reactivation levels induced by sera from FMDV-infected and non-infected or vaccinated pigs. These results confirm the high-throughput, multiscreening potential of these recombinant ßgalactosidase to act as promising diagnostic tools based on biosensors.