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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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In vivo growth kinetics of rTGEV-Δ7 virus.(A) Two- to three-day-old piglets were inoculated with 1×107 pfu/pig of rTGEV-wt and rTGEV-Δ7 viruses by two routes (oral and nasal) in combination. At 0.5, 1, 2, 3, 4 and 5 days post inoculation two animals per group were sacrificed, and the lungs were harvested. rTGEV-wt (blue) and rTGEV-Δ7 (red), recovered from lung, were titrated. Triangles indicated sentinel animals. (B) Two- to three-day-old piglets were inoculated with 1×107 pfu/pig of rTGEV-SC11-wt and rTGEV-SC11-Δ7 viruses by three routes (oral, intranasal and intragastric) in combination. At indicated days post inoculation two animals per group were sacrificed, and the lung and the gut were harvested. rTGEV-SC11-wt (blue) and rTGEV-SC11-Δ7 (red) titers in gut are represented. Triangles indicate sentinel animals. Error bars indicate the standard deviation from three independent experiments.
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ppat-1002090-g012: In vivo growth kinetics of rTGEV-Δ7 virus.(A) Two- to three-day-old piglets were inoculated with 1×107 pfu/pig of rTGEV-wt and rTGEV-Δ7 viruses by two routes (oral and nasal) in combination. At 0.5, 1, 2, 3, 4 and 5 days post inoculation two animals per group were sacrificed, and the lungs were harvested. rTGEV-wt (blue) and rTGEV-Δ7 (red), recovered from lung, were titrated. Triangles indicated sentinel animals. (B) Two- to three-day-old piglets were inoculated with 1×107 pfu/pig of rTGEV-SC11-wt and rTGEV-SC11-Δ7 viruses by three routes (oral, intranasal and intragastric) in combination. At indicated days post inoculation two animals per group were sacrificed, and the lung and the gut were harvested. rTGEV-SC11-wt (blue) and rTGEV-SC11-Δ7 (red) titers in gut are represented. Triangles indicate sentinel animals. Error bars indicate the standard deviation from three independent experiments.

Mentions: Newborn piglets were infected with rTGEV-wt and rTGEV-Δ7 viruses. Both viruses showed similar growth kinetics in the lung, although gene 7 deletion mutant virus reached higher titers than the parental virus at early times post infection (Figure 12A). Virulent TGEV strains replicate in the villious epithelial cells of the small intestine and in lung cells, causing severe diarrhea in newborn piglets [57], [114], [115]. The respiratory and enteric tropism of the rTGEVs can be modified by the introduction of an S gene from a virulent strain [57], [114], [115]. The rTGEV-Δ7 deletion mutant used throughout this paper was generated with an exclusively respiratory tropism (see Materials and Methods). To study the relevance of protein 7 in a virulent virus, a recombinant virus with respiratory and enteric tropism, lacking the expression of the gene 7 (rTGEV-SC11-Δ7) was engineered [57]. Growth in lung of rTGEV-SC11-wt and rTGEV-SC11-Δ7 viruses was similar to that of the previous mutant and wild-type viruses (data not shown). Interestingly, the rTGEV-SC11-Δ7 showed accelerated growth kinetics in gut, compared to the wild-type virus (Figure 12B). This behavior correlated with more pronounced clinical symptoms (Figure S5A). Both rTGEV-SC11-wt and rTGEV-SC11-Δ7 infected animals had the same final survival ratio (50%) (Figure S5B). Nevertheless, animals infected with rTGEV-SC11-Δ7 died six days before that those infected with rTGEV-SC11-wt (Figure S5B). Accordingly, virus was detected only in sentinel animals in contact with rTGEV-wt infected piglets, but not in those in close proximity to the rTGEV-SC11-Δ7 infected animals (Figure 12B). This result suggested that the presence of protein 7 facilitated animal survival and virus shedding.


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)

In vivo growth kinetics of rTGEV-Δ7 virus.(A) Two- to three-day-old piglets were inoculated with 1×107 pfu/pig of rTGEV-wt and rTGEV-Δ7 viruses by two routes (oral and nasal) in combination. At 0.5, 1, 2, 3, 4 and 5 days post inoculation two animals per group were sacrificed, and the lungs were harvested. rTGEV-wt (blue) and rTGEV-Δ7 (red), recovered from lung, were titrated. Triangles indicated sentinel animals. (B) Two- to three-day-old piglets were inoculated with 1×107 pfu/pig of rTGEV-SC11-wt and rTGEV-SC11-Δ7 viruses by three routes (oral, intranasal and intragastric) in combination. At indicated days post inoculation two animals per group were sacrificed, and the lung and the gut were harvested. rTGEV-SC11-wt (blue) and rTGEV-SC11-Δ7 (red) titers in gut are represented. Triangles indicate sentinel animals. Error bars indicate the standard deviation from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002090-g012: In vivo growth kinetics of rTGEV-Δ7 virus.(A) Two- to three-day-old piglets were inoculated with 1×107 pfu/pig of rTGEV-wt and rTGEV-Δ7 viruses by two routes (oral and nasal) in combination. At 0.5, 1, 2, 3, 4 and 5 days post inoculation two animals per group were sacrificed, and the lungs were harvested. rTGEV-wt (blue) and rTGEV-Δ7 (red), recovered from lung, were titrated. Triangles indicated sentinel animals. (B) Two- to three-day-old piglets were inoculated with 1×107 pfu/pig of rTGEV-SC11-wt and rTGEV-SC11-Δ7 viruses by three routes (oral, intranasal and intragastric) in combination. At indicated days post inoculation two animals per group were sacrificed, and the lung and the gut were harvested. rTGEV-SC11-wt (blue) and rTGEV-SC11-Δ7 (red) titers in gut are represented. Triangles indicate sentinel animals. Error bars indicate the standard deviation from three independent experiments.
Mentions: Newborn piglets were infected with rTGEV-wt and rTGEV-Δ7 viruses. Both viruses showed similar growth kinetics in the lung, although gene 7 deletion mutant virus reached higher titers than the parental virus at early times post infection (Figure 12A). Virulent TGEV strains replicate in the villious epithelial cells of the small intestine and in lung cells, causing severe diarrhea in newborn piglets [57], [114], [115]. The respiratory and enteric tropism of the rTGEVs can be modified by the introduction of an S gene from a virulent strain [57], [114], [115]. The rTGEV-Δ7 deletion mutant used throughout this paper was generated with an exclusively respiratory tropism (see Materials and Methods). To study the relevance of protein 7 in a virulent virus, a recombinant virus with respiratory and enteric tropism, lacking the expression of the gene 7 (rTGEV-SC11-Δ7) was engineered [57]. Growth in lung of rTGEV-SC11-wt and rTGEV-SC11-Δ7 viruses was similar to that of the previous mutant and wild-type viruses (data not shown). Interestingly, the rTGEV-SC11-Δ7 showed accelerated growth kinetics in gut, compared to the wild-type virus (Figure 12B). This behavior correlated with more pronounced clinical symptoms (Figure S5A). Both rTGEV-SC11-wt and rTGEV-SC11-Δ7 infected animals had the same final survival ratio (50%) (Figure S5B). Nevertheless, animals infected with rTGEV-SC11-Δ7 died six days before that those infected with rTGEV-SC11-wt (Figure S5B). Accordingly, virus was detected only in sentinel animals in contact with rTGEV-wt infected piglets, but not in those in close proximity to the rTGEV-SC11-Δ7 infected animals (Figure 12B). This result suggested that the presence of protein 7 facilitated animal survival and virus shedding.

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