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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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Apoptosis caused by rTGEV-Δ7.(A) Apoptosis levels in mock, rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) infected cells were evaluated at 4, 8 and 12 hpi, by flow cytometry. Annexin V-PI double staining was performed to differentiate cells in early apoptosis (Annexin V+, PI−) from those in late apoptosis (Annexin V+, PI+) stages. (B) Detection of active caspase 3 by Western-blot. Total protein was extracted from ST cells infected with rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) viruses, at the indicated times post infection. Active caspase 3 was detected using specific antibodies for the cleaved form. β-actin was detected as a loading control.
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ppat-1002090-g003: Apoptosis caused by rTGEV-Δ7.(A) Apoptosis levels in mock, rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) infected cells were evaluated at 4, 8 and 12 hpi, by flow cytometry. Annexin V-PI double staining was performed to differentiate cells in early apoptosis (Annexin V+, PI−) from those in late apoptosis (Annexin V+, PI+) stages. (B) Detection of active caspase 3 by Western-blot. Total protein was extracted from ST cells infected with rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) viruses, at the indicated times post infection. Active caspase 3 was detected using specific antibodies for the cleaved form. β-actin was detected as a loading control.

Mentions: The main cause of the cytopathic effect induced by TGEV infection is apoptosis programmed cell death [63], [64], [65]. To analyze whether the increased cell death during rTGEV-Δ7 infection was due to an enhanced apoptosis, cells infected either with rTGEV-wt or rTGEV-Δ7 were simultaneously stained with PI and Annexin V, and monitored by flow cytometry. Mock infected cells remained viable (Annexin V−, PI−) throughout the experiment, indicating that the treatment did not induce apoptosis by itself (Figure 3A). As expected, the wild-type virus infection induced apoptosis (Annexin V+), and a cell population in late apoptosis (Annexin V+, PI+) was evident at 12 hpi (Figure 3A). Mutant rTGEV-Δ7 also triggered apoptosis but faster and stronger than that caused by the rTGEV-wt virus, with a 2-fold increase in apoptotic cells at 8 hpi and only 36% live cells at 12 hpi (Figure 3A).


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)

Apoptosis caused by rTGEV-Δ7.(A) Apoptosis levels in mock, rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) infected cells were evaluated at 4, 8 and 12 hpi, by flow cytometry. Annexin V-PI double staining was performed to differentiate cells in early apoptosis (Annexin V+, PI−) from those in late apoptosis (Annexin V+, PI+) stages. (B) Detection of active caspase 3 by Western-blot. Total protein was extracted from ST cells infected with rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) viruses, at the indicated times post infection. Active caspase 3 was detected using specific antibodies for the cleaved form. β-actin was detected as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002090-g003: Apoptosis caused by rTGEV-Δ7.(A) Apoptosis levels in mock, rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) infected cells were evaluated at 4, 8 and 12 hpi, by flow cytometry. Annexin V-PI double staining was performed to differentiate cells in early apoptosis (Annexin V+, PI−) from those in late apoptosis (Annexin V+, PI+) stages. (B) Detection of active caspase 3 by Western-blot. Total protein was extracted from ST cells infected with rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) viruses, at the indicated times post infection. Active caspase 3 was detected using specific antibodies for the cleaved form. β-actin was detected as a loading control.
Mentions: The main cause of the cytopathic effect induced by TGEV infection is apoptosis programmed cell death [63], [64], [65]. To analyze whether the increased cell death during rTGEV-Δ7 infection was due to an enhanced apoptosis, cells infected either with rTGEV-wt or rTGEV-Δ7 were simultaneously stained with PI and Annexin V, and monitored by flow cytometry. Mock infected cells remained viable (Annexin V−, PI−) throughout the experiment, indicating that the treatment did not induce apoptosis by itself (Figure 3A). As expected, the wild-type virus infection induced apoptosis (Annexin V+), and a cell population in late apoptosis (Annexin V+, PI+) was evident at 12 hpi (Figure 3A). Mutant rTGEV-Δ7 also triggered apoptosis but faster and stronger than that caused by the rTGEV-wt virus, with a 2-fold increase in apoptotic cells at 8 hpi and only 36% live cells at 12 hpi (Figure 3A).

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