Análisis de la interacción entre el virus de la gripe y la respuesta celular innata
- Autores: María Teresa Pérez Cidoncha
- Directores de la Tesis: Juan Ortín Montón (dir. tes.)
- Lectura: En la Universidad Autónoma de Madrid ( España ) en 2013
- Idioma: español
- Tribunal Calificador de la Tesis: Esteban Domingo Solans (presid.), Juan Antonio García Alvarez (secret.), Estanislao Nistal Villán (voc.), José Antonio Melero Fondevila (voc.), Purificación Fortes (voc.)
- Calificación: Apto Cum Laude
- Materias:
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- Resumen
As a consequence of influenza virus infection, the cell fires an innate immune response to restrict the multiplication of the virus. In general, viruses have evolved a variety of countermeasures to avoid this response and maintain reasonable replication efficiency. The influenza A viruses counteract the cell innate immune response at several steps, including blocking the RIG I-dependent activation of interferon (IFN) transcription, the IFN-dependent activation of IFN-stimulated genes (ISGs) and the activity of various ISGs. The multifunctional NS1 protein provides most of the influenza virus modulation of the IFN response. In addition to NS1, in the last years other influenza virus genes have been reported to alter the cellular innate immune response.
To understand the role of NS1 in this counteraction, we introduced random mutations in NS segment.
Using this strategy, we have obtained a population of 30.000-40.000 different mutant viruses and selected those able to induce IFN expression and to grow efficiently. The sequence analysis of the selected virus clones showed that non-synonymous mutations occurred at many phylogenetically conserved positions within NS1 protein, suggesting that they could be relevant for the mutant phenotype. Most of these mutant viruses showed normal replication kinetics and high titres and all mutants induced an IFN response stronger than wt virus. Surprisingly, one of the mutants led to an antiviral activity very similar to the one obtained with a mutant virus lacking NS1, although it contains a single aminoacid substitution.
To determine whether other viral genes are also important in the interplay between the virus and the host IFN response we have undertaken a non-biassed genetic approach. We carried out serial passage of wt virus in IFN non-responsive cells and selected for viruses that were able to induce IFN. We reasoned that, by replication in the absence of the IFN selection pressure the virus could mutate at positions normally restricted and could find new optimal sequence solutions. Deep sequencing of selected virus populations and individual virus mutants indicated that non-synonymous mutations occurred at many phylogenetically conserved positions in all virus segments. Several of these mutations are recurrent in independently evolved virus populations and also in individual mutants. Only one of the six individual virus mutants studied contained a mutation in NS1, although all of them induced IFN and ISGs and were unable to counteract added IFN. Several virus mutants accumulated large amounts of large internal deletions in several virus genes but nonetheless replicated to high titres and showed mutations in the M1/M2 proteins. These results suggest that essentially the complete virus gene complement is adapted to counteract the IFN response and drift away of such an optimal sequence when the IFN response is ablated.
In summary both approaches are complementary and allowed us to obtain a general map of the mechanisms exploited by the virus to counteract the IFN response. The results support the use of these strategies for the generation and identification of viral mutants affected in IFN counteraction.
