Tesis doctoral de Jazmina Libertad González Cruz
Transmissible gastroenteritis virus (tgev) genome contains three accessory genes: 3a, 3b and 7. Gene 7 is only present in members of the species alphacoronavirus 1, and encodes a hydrophobic protein of 78 aa. To study gene 7 function, a recombinant tgev virus lacking gene 7 was engineered (rtgev-¿7). Both the deletion mutant and the parental (rtgev-wt) viruses showed the same growth and viral rna synthesis kinetics in tissue culture. Nevertheless, cells infected with rtgev-¿7 virus showed an increased cytopathic effect caused by an enhanced apoptosis mediated by caspase activation. analysis of macromolecular synthesis showed that rtgev-¿7 virus infection led to host translational shut-off and increased cellular rna degradation compared with rtgev-wt infection. An increase of eukaryotic translation initiation factor 2 (eif2¿) phosphorylation and an enhanced nuclease activity were observed in rtgev-¿7 virus infected cells. These results suggested that the removal of gene 7 promoted an intensified dsrna-activated host antiviral response. a conserved sequence motif that potentially mediates binding to protein phosphatase 1 catalytic subunit (pp1c), a key regulator of the cell antiviral defenses, was identified in protein 7 by bioinformatic analysis. In fact, pull-down assays demonstrated the interaction between pp1 and tgev protein 7, but not a protein 7 mutant lacking pp1c binding motif. Furthermore, eif2¿ was also present in this complex, strongly suggesting a regulation of eif2¿ phosphorylation by protein 7. Moreover, the interaction between protein 7 and pp1 was required for eif2a dephosphotylation and inhibition of cell rna degradation. These data indicated that tgev protein 7 counteracted host antiviral response by its association with pp1c. the analysis of the gene expression profiling in rtgev-wt and rtgev-¿7 infected cells using microarrays, showed that the genes differentially expressed were involved in response to virus and inflammation. These results were confirmed by rt-qpcr using specific taqman assays, suggesting that the absence of protein 7 during tgev infection led to enhanced levels of pro-inflammatory cytokines such as ifníY, tnf, rantes, ccl2 and ccl4. inoculation of newborn piglets with rtgev-¿7 and rtgev-wt viruses showed that rtgev-¿7 virus led to an accelerated growth kinetics and pathology compared with the parental virus. Furthermore, the increased pathology caused by the rtgev-¿7 infection could be due to and exacerbate macrophage activation, as a higher macrophage resumen en inglés 2 infiltration in lung was observed in the animals infected with the deletion mutant virus compared with those infected with the wild type. overall, the results indicated that gene 7 counteracted host cell defenses, and modified tgev virulence increasing tgev survival. Therefore, the acquisition of gene 7 by the tgev genome most likely has provided a selective advantage to the virus.
Datos académicos de la tesis doctoral ««el gen 7 de alfacoronavirus 1 contrarresta a la respuesta huésped y modula la virulencia del virus».«
- Título de la tesis: «el gen 7 de alfacoronavirus 1 contrarresta a la respuesta huésped y modula la virulencia del virus».
- Autor: Jazmina Libertad González Cruz
- Universidad: Autónoma de Madrid
- Fecha de lectura de la tesis: 25/11/2011
Dirección y tribunal
- Director de la tesis
- Luis EnJuan es Sanchez
- Tribunal
- Presidente del tribunal: María no Esteban rodriguez
- José Antonio Melero fondevilla (vocal)
- Francisco Javier Ortego alonso (vocal)
- isidoro González martínez (vocal)