Estudio de los complejos replicativos del torovirus equino Bev

• Autores: Ginés Francisco Avila Pérez
• Directores de la Tesis: María Dolores Rodríguez Aguirre (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.), Fernando Almazán Toral (secret.), Covadonga Alonso Martí (voc.), Pablo Gastaminza Landart (voc.), Francisco Javier Ortego Alonso (voc.)
• Materias:
  • Ciencias de la vida
    • Virología
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Plus-stranded RNA viruses replicate in the cytosol of infected cells, in membrane-bound replication complexes containing the replicase proteins, the viral RNA and host proteins. The formation of the replication and transcription complexes (RTCs) through the rearrangement of cellular membranes is currently being actively studied for viruses belonging to different viral families. In this work we identified double membrane vesicles (DMVs) in the cytoplasm of cells infected with the equine torovirus Berne virus (BEV), the prototype member of the Torovirus genus (Coronaviridae Family, Nidovirales Order). Using confocal microscopy and transmission electron microscopy we observed a close relationship between the RTCs and the DMVs of BEV. The examination of BEV infected cells revealed that the replicase proteins colocalized with each other and with newly synthetized RNA, and were associated to the membrane rearrangement induced by BEV. However, the dsRNA, an intermediate of viral replication, was exclusively limited to the interior of DMVs. Also, an in-depth ultrastructural analysis of cells infected with BEV was performed to characterize the architecture of torovirus replication factories and to learn about their biogenesis during the infection. Analysis by conventional transmission electron microscopy suggested that the DMVs form a reticulovesicular network (RVN) resembling those described for the related severe acute respiratory syndrome coronavirus (SARS-CoV) and the equine arteritis virus (EAV). Using serial sectioning and electron tomography we confirmed the formation of a RVN in BEV infected cells, where the DMVs outer membranes are interconnected with each other and with the ER. Like in EAV, convoluted membranes were not observed in the RVNs. However, we observed paired membranes lacking luminal space, which are connected with the DMVs, and likely represent early structures that will evolve to give rise to DMVs. Interestingly, curled membranes resembling the spherules described in IBV were observed at late time post-infection in BEV-infected cells, however, after careful examination of the tomograms we concluded that they are not true spherules, but aberrant structures arising late on the DMV formation. This work complements the knowledge about the membrane rearrangements promoted by nidovirus infection, and all this taken together indicates that the nidoviruses use a similar strategy to establish their membranous replication factories.

    On the other hand, recent studies suggest that some positive-stranded RNA viruses exploit the autophagy pathway to facilitate their own replication. We demonstrated that BEV induces autophagy, with an active flux, in the infected cells at late times post-infection. However, the autophagy induction by nutrient deprivation, or its inhibition by pharmacological treatment with inhibitors of autophagosome formation or agents that block the autophagosome degradation, does not produce changes in BEV replication. In the same way, the replication of BEV was not affected in autophagy deficient cells lacking Beclin 1 or LC3B. Overall, our results suggest that the autophagy pathway is activated during BEV infection at late time post-infection, but this activation is not necessary for the viral replication.