Coronavirus Gene 7 Counteracts Host Defenses and Modulates Virus Virulence

Abstract

Transmissible gastroenteritis virus (TGEV) genome contains three accessory genes: 3a, 3b and 7. Gene 7 is only present in members of coronavirus genus a1, and encodes a hydrophobic protein of 78 aa. To study gene 7 function, a recombinant TGEV virus lacking gene 7 was engineered (rTGEV-Δ7). Both the mutant and the parental (rTGEV- wt) viruses showed the same growth and viral RNA accumulation kinetics in tissue cultures. Nevertheless, cells infected with rTGEV-Δ7 virus showed an increased cytopathic effect caused by an enhanced apoptosis mediated by caspase activation. Macromolecular synthesis analysis showed that rTGEV-Δ7 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 (eIF2α) phosphorylation and an enhanced nuclease, most likely RNase L, activity were observed in rTGEV-Δ7 virus infected cells. These results suggested that the removal of gene 7 promoted an intensified dsRNA-activated host antiviral response. In protein 7 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. We postulated that TGEV protein 7 may counteract host antiviral response by its association with PP1c. In fact, pull-down assays demonstrated the interaction between TGEV protein 7, but not a protein 7 mutant lacking PP1c binding motif, with PP1. Moreover, the interaction between protein 7 and PP1 was required, during the infection, for eIF2α dephosphorylation and inhibition of cell RNA degradation. Inoculation of newborn piglets with rTGEV-Δ7 and rTGEV- wt viruses showed that rTGEV-Δ7 virus presented accelerated growth kinetics and pathology compared with the parental virus. Overall, the results indicated that gene 7 counteracted host cell defenses, and modified TGEV persistence increasing TGEV survival. Therefore, the acquisition of gene 7 by the TGEV genome most likely has provided a selective advantage to the virus.

Author Summary

Innate immune response is the first line of antiviral defense. Viruses have developed diverse strategies to evade this deleterious response, ensuring their survival. Several CoV accessory genes play a central role in these pathways. Nevertheless, the molecular mechanisms by which they exert their function are still unknown. The generation of a rTGEV without gene 7 expression allowed us to study the role of protein 7 in the modulation of the antiviral response. The absence of protein 7 during TGEV infection caused an enhanced apoptosis and a translational shutoff, due to an increased cellular RNA degradation and eIF2α phosphorylation. We identified a protein phosphatase 1 (PP1) binding motif in protein 7, and a TGEV protein 7-PP1 interaction was demonstrated. We propose a novel mechanism to counteract dsRNA-induced antiviral response by RNA viruses. In vitro results were in agreement with the enhanced virulence of the gene 7 deletion mutant virus in infected piglets. Our results demonstrated that protein 7 modifies TGEV virulence, reducing virus pathology and increasing the period of virus shedding. This effect also benefits the host decreasing clinical disease and extending its survival. These observations could justify the incorporation and maintenance of gene 7 to genus a1 CoVs during their evolution.