El gen 7 de alfacoronavirus 1 contrarresta a la respuesta huésped y modula la virulencia del virus"

Résumé

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 IFNß, 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.