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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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Complementation of rTGEV-Δ7 produced apoptosis and RNA degradation by protein 7 provided in trans.Generation of ST cells expressing TGEV protein 7 in trans. (A) Scheme of TGEV protein 7 expressed by the gene transfected into ST cells. Hemaglutinin tag (HA, light blue) was inserted after signal peptide (blue). (B) Protein 7 expression levels for the three ST-HA-7 selected cellular clones (C1, C2 and C3), were analyzed by immunofluorescence (left). Tagged protein 7 was detected with an anti-HA antibody stained in green, and cell nucleus were stained in blue. Percentage of HA-7 expressing cells is indicated. HA-7 protein accumulation was evaluated by Western-blot (right). HA-7 band is indicated, and corresponds to tagged protein cleaved form (7 KDa). (C) ST cells, or ST cells expressing HA-tagged protein 7 (ST-HA-7) were used to analyze apoptosis levels by flow cytometry. Apoptosis levels in mock, rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) infected cells were evaluated at 12 hpi. 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. (D) ST cells and the three ST-HA-7 cell clones obtained were mock, rTGEV-wt or rTGEV-Δ7 infected. Total RNA was extracted at 18 hpi. Cellular RNA integrity was analyzed using a Bioanalyzer. 28S and 18S rRNAs are indicated on the right.
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ppat-1002090-g009: Complementation of rTGEV-Δ7 produced apoptosis and RNA degradation by protein 7 provided in trans.Generation of ST cells expressing TGEV protein 7 in trans. (A) Scheme of TGEV protein 7 expressed by the gene transfected into ST cells. Hemaglutinin tag (HA, light blue) was inserted after signal peptide (blue). (B) Protein 7 expression levels for the three ST-HA-7 selected cellular clones (C1, C2 and C3), were analyzed by immunofluorescence (left). Tagged protein 7 was detected with an anti-HA antibody stained in green, and cell nucleus were stained in blue. Percentage of HA-7 expressing cells is indicated. HA-7 protein accumulation was evaluated by Western-blot (right). HA-7 band is indicated, and corresponds to tagged protein cleaved form (7 KDa). (C) ST cells, or ST cells expressing HA-tagged protein 7 (ST-HA-7) were used to analyze apoptosis levels by flow cytometry. Apoptosis levels in mock, rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) infected cells were evaluated at 12 hpi. 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. (D) ST cells and the three ST-HA-7 cell clones obtained were mock, rTGEV-wt or rTGEV-Δ7 infected. Total RNA was extracted at 18 hpi. Cellular RNA integrity was analyzed using a Bioanalyzer. 28S and 18S rRNAs are indicated on the right.

Mentions: To assess whether the absence of protein 7 during TGEV infection was responsible for the observed phenotype, ST cells stably expressing TGEV protein 7 (ST-HA-7) were generated. In order to detect protein 7, a hemagglutinin (HA) tag was inserted between the signal peptide and the rest of the protein (Figure 9A). Protein 7 expression was confirmed by immunofluorescence and Western-blot analysis (Figure 9B). Three ST-HA-7 cellular clones (C1, C2 and C3), with different protein 7 expression levels were selected (Figure 9B). The effect of protein 7 provided in trans on apoptosis and cellular RNA degradation was analyzed. Infection of ST cells by rTGEV-Δ7 caused a stronger apoptosis than the rTGEV-wt virus, as previously observed (Figure 9C). Protein 7 provided in trans significantly reduced apoptosis both in rTGEV-Δ7 infected cells and in rTGEV-wt infected ones (Figure 9C). Moreover, infection of ST cells by rTGEV-Δ7 caused higher RNA degradation than the rTGEV-wt virus, as previously described (Figure 9D). The amount of protein 7 directly correlated with the inhibition of RNA degradation, suggesting that protein 7 expression in trans prevented nuclease activation (Figure 9D). Furthermore, GADD34 mRNA expression (Figure S2A) and eIF2α phosphorylation levels (Figure S2B) were reduced by protein 7 expression in trans. Altogether, these results demonstrated that the specific phenotype of the rTGEV-Δ7 virus was due to TGEV protein 7 absence, as it was reverted to the rTGEV-wt phenotype, in a dose-dependent manner, by providing protein 7 in trans.


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)

Complementation of rTGEV-Δ7 produced apoptosis and RNA degradation by protein 7 provided in trans.Generation of ST cells expressing TGEV protein 7 in trans. (A) Scheme of TGEV protein 7 expressed by the gene transfected into ST cells. Hemaglutinin tag (HA, light blue) was inserted after signal peptide (blue). (B) Protein 7 expression levels for the three ST-HA-7 selected cellular clones (C1, C2 and C3), were analyzed by immunofluorescence (left). Tagged protein 7 was detected with an anti-HA antibody stained in green, and cell nucleus were stained in blue. Percentage of HA-7 expressing cells is indicated. HA-7 protein accumulation was evaluated by Western-blot (right). HA-7 band is indicated, and corresponds to tagged protein cleaved form (7 KDa). (C) ST cells, or ST cells expressing HA-tagged protein 7 (ST-HA-7) were used to analyze apoptosis levels by flow cytometry. Apoptosis levels in mock, rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) infected cells were evaluated at 12 hpi. 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. (D) ST cells and the three ST-HA-7 cell clones obtained were mock, rTGEV-wt or rTGEV-Δ7 infected. Total RNA was extracted at 18 hpi. Cellular RNA integrity was analyzed using a Bioanalyzer. 28S and 18S rRNAs are indicated on the right.
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Related In: Results  -  Collection

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

ppat-1002090-g009: Complementation of rTGEV-Δ7 produced apoptosis and RNA degradation by protein 7 provided in trans.Generation of ST cells expressing TGEV protein 7 in trans. (A) Scheme of TGEV protein 7 expressed by the gene transfected into ST cells. Hemaglutinin tag (HA, light blue) was inserted after signal peptide (blue). (B) Protein 7 expression levels for the three ST-HA-7 selected cellular clones (C1, C2 and C3), were analyzed by immunofluorescence (left). Tagged protein 7 was detected with an anti-HA antibody stained in green, and cell nucleus were stained in blue. Percentage of HA-7 expressing cells is indicated. HA-7 protein accumulation was evaluated by Western-blot (right). HA-7 band is indicated, and corresponds to tagged protein cleaved form (7 KDa). (C) ST cells, or ST cells expressing HA-tagged protein 7 (ST-HA-7) were used to analyze apoptosis levels by flow cytometry. Apoptosis levels in mock, rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) infected cells were evaluated at 12 hpi. 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. (D) ST cells and the three ST-HA-7 cell clones obtained were mock, rTGEV-wt or rTGEV-Δ7 infected. Total RNA was extracted at 18 hpi. Cellular RNA integrity was analyzed using a Bioanalyzer. 28S and 18S rRNAs are indicated on the right.
Mentions: To assess whether the absence of protein 7 during TGEV infection was responsible for the observed phenotype, ST cells stably expressing TGEV protein 7 (ST-HA-7) were generated. In order to detect protein 7, a hemagglutinin (HA) tag was inserted between the signal peptide and the rest of the protein (Figure 9A). Protein 7 expression was confirmed by immunofluorescence and Western-blot analysis (Figure 9B). Three ST-HA-7 cellular clones (C1, C2 and C3), with different protein 7 expression levels were selected (Figure 9B). The effect of protein 7 provided in trans on apoptosis and cellular RNA degradation was analyzed. Infection of ST cells by rTGEV-Δ7 caused a stronger apoptosis than the rTGEV-wt virus, as previously observed (Figure 9C). Protein 7 provided in trans significantly reduced apoptosis both in rTGEV-Δ7 infected cells and in rTGEV-wt infected ones (Figure 9C). Moreover, infection of ST cells by rTGEV-Δ7 caused higher RNA degradation than the rTGEV-wt virus, as previously described (Figure 9D). The amount of protein 7 directly correlated with the inhibition of RNA degradation, suggesting that protein 7 expression in trans prevented nuclease activation (Figure 9D). Furthermore, GADD34 mRNA expression (Figure S2A) and eIF2α phosphorylation levels (Figure S2B) were reduced by protein 7 expression in trans. Altogether, these results demonstrated that the specific phenotype of the rTGEV-Δ7 virus was due to TGEV protein 7 absence, as it was reverted to the rTGEV-wt phenotype, in a dose-dependent manner, by providing protein 7 in trans.

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