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Coronavirus gene 7 counteracts host defenses and modulates virus virulence.

Cruz JL, Sola I, Becares M, Alberca B, Plana J, Enjuanes L, Zuñiga S - PLoS Pathog. (2011)

Bottom Line: 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.These results suggested that the removal of gene 7 promoted an intensified dsRNA-activated host antiviral response.Overall, the results indicated that gene 7 counteracted host cell defenses, and modified TGEV persistence increasing TGEV survival.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Biotecnología, CNB, CSIC, Department of Molecular and Cell Biology, Darwin 3, Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.

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.

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eIF2α phosphorylation during rTGEV infection.(A) Total protein was extracted, at indicated times post infection, from ST cells infected at a moi of 5 with rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) viruses. Accumulation of total eIF2α and phosphorylated eIF2α (eIF2α-P), was analyzed by Western-blot. (B) eIF2α and eIF2α-P amounts were estimated by densitometric analysis. The graph represented eIF2α/eIF2α-P ratio in mock (green), rTGEV-wt (blue) and rTGEV-Δ7 (red) infected cells at indicated hpi. Error bars indicate the standard deviation from six independent experiments. r.u., relative units. *, p-value <0.05; **, p-value <0.01.
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ppat-1002090-g008: eIF2α phosphorylation during rTGEV infection.(A) Total protein was extracted, at indicated times post infection, from ST cells infected at a moi of 5 with rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) viruses. Accumulation of total eIF2α and phosphorylated eIF2α (eIF2α-P), was analyzed by Western-blot. (B) eIF2α and eIF2α-P amounts were estimated by densitometric analysis. The graph represented eIF2α/eIF2α-P ratio in mock (green), rTGEV-wt (blue) and rTGEV-Δ7 (red) infected cells at indicated hpi. Error bars indicate the standard deviation from six independent experiments. r.u., relative units. *, p-value <0.05; **, p-value <0.01.

Mentions: Protein synthesis is frequently reduced when cells are under stress, such as that caused by virus infection, by increasing the phosphorylation levels of the eIF2α subunit at serine 51 [89]. eIF2α phosphorylation, during rTGEV infection, was analyzed by Western-blot using antibodies specific for the phosphorylated (eIF2α-P) and total forms of this factor, respectively. Wild-type infection increased eIF2α-P levels (Figure 8A), reaching a maximum at 8 hpi (Figure 8B). As previously described, for other stress conditions, eIF2α-P levels decreased at late times post-infection [90], [91]. Similarly, rTGEV-Δ7 infection also induced eIF2α phosphorylation (Figure 8A) but to significantly higher levels than those observed during rTGEV-wt infection (Figures 8A and 8B). Interestingly, the highest difference was detected at 10 hpi, concomitantly with the time at which the mutant virus induced the translational shutoff (Figure 8B). The increased eIF2α phosphorylation was maintained, although at different levels, from 8hpi to 10 hpi, what could be sufficient to account for the translational shutoff, according to previously published studies [92], [93]. Altogether, this result indicated that, besides cellular RNA degradation, rTGEV-Δ7-induced translational shutoff is probably due to an increased and sustained eIF2α phosphorylation.


Coronavirus gene 7 counteracts host defenses and modulates virus virulence.

Cruz JL, Sola I, Becares M, Alberca B, Plana J, Enjuanes L, Zuñiga S - PLoS Pathog. (2011)

eIF2α phosphorylation during rTGEV infection.(A) Total protein was extracted, at indicated times post infection, from ST cells infected at a moi of 5 with rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) viruses. Accumulation of total eIF2α and phosphorylated eIF2α (eIF2α-P), was analyzed by Western-blot. (B) eIF2α and eIF2α-P amounts were estimated by densitometric analysis. The graph represented eIF2α/eIF2α-P ratio in mock (green), rTGEV-wt (blue) and rTGEV-Δ7 (red) infected cells at indicated hpi. Error bars indicate the standard deviation from six independent experiments. r.u., relative units. *, p-value <0.05; **, p-value <0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111541&req=5

ppat-1002090-g008: eIF2α phosphorylation during rTGEV infection.(A) Total protein was extracted, at indicated times post infection, from ST cells infected at a moi of 5 with rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) viruses. Accumulation of total eIF2α and phosphorylated eIF2α (eIF2α-P), was analyzed by Western-blot. (B) eIF2α and eIF2α-P amounts were estimated by densitometric analysis. The graph represented eIF2α/eIF2α-P ratio in mock (green), rTGEV-wt (blue) and rTGEV-Δ7 (red) infected cells at indicated hpi. Error bars indicate the standard deviation from six independent experiments. r.u., relative units. *, p-value <0.05; **, p-value <0.01.
Mentions: Protein synthesis is frequently reduced when cells are under stress, such as that caused by virus infection, by increasing the phosphorylation levels of the eIF2α subunit at serine 51 [89]. eIF2α phosphorylation, during rTGEV infection, was analyzed by Western-blot using antibodies specific for the phosphorylated (eIF2α-P) and total forms of this factor, respectively. Wild-type infection increased eIF2α-P levels (Figure 8A), reaching a maximum at 8 hpi (Figure 8B). As previously described, for other stress conditions, eIF2α-P levels decreased at late times post-infection [90], [91]. Similarly, rTGEV-Δ7 infection also induced eIF2α phosphorylation (Figure 8A) but to significantly higher levels than those observed during rTGEV-wt infection (Figures 8A and 8B). Interestingly, the highest difference was detected at 10 hpi, concomitantly with the time at which the mutant virus induced the translational shutoff (Figure 8B). The increased eIF2α phosphorylation was maintained, although at different levels, from 8hpi to 10 hpi, what could be sufficient to account for the translational shutoff, according to previously published studies [92], [93]. Altogether, this result indicated that, besides cellular RNA degradation, rTGEV-Δ7-induced translational shutoff is probably due to an increased and sustained eIF2α phosphorylation.

Bottom Line: 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.These results suggested that the removal of gene 7 promoted an intensified dsRNA-activated host antiviral response.Overall, the results indicated that gene 7 counteracted host cell defenses, and modified TGEV persistence increasing TGEV survival.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Biotecnología, CNB, CSIC, Department of Molecular and Cell Biology, Darwin 3, Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.

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.

Show MeSH
Related in: MedlinePlus