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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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Generation of a recombinant TGEV virus lacking protein 7 expression (rTGEV-Δ7).(A) Genus α1 CoV protein 7a sequence alignment, using T-COFFEE [135]. Protein 7a sequences from the canine (CCoV) and porcine respiratory (PRCV) CoVs, transmissible gastroenteritis (TGEV) and feline infectious peritonitis (FIPV) viruses were used. GenBank accession numbers are ADB28914.1, ABG89313.1, CAA80842.1, and CAA62190.1, respectively. In silico prediction of TGEV protein 7 domains is represented. Transmembrane domains (TM) are in green [PredictProtein, [136]], the signal peptide in blue [Signal P3.0 Server, [137]], and a conserved phosphorilable Serine in red (S-Phos) [NetPhos 2.0 Server, [138]]. The predicted topology of TGEV protein 7 is also represented in lower panel [PSORTII [139]]. Signal peptide cleavage is indicated by a red arrowhead. S-Phos is indicated by a red star. (B) Mutations introduced to generate a rTGEV-Δ7 virus, right panel. The scheme of TGEV gRNA is shown in the upper part. The white letters represent the CS. Nucleotide change is indicated with a blue square, and the deletion (Δ) as a white square. Northern blot of subgenomic mRNAs (sgmRNAs) produced during rTGEV infections, right panel. ST cells were infected with rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) viruses, at a moi of 5. Total RNA was extracted at 8 hpi. The sgmRNAs for the spike (S), 3a, envelope (E), membrane (M), nucleocapsid (N) proteins, and protein 7 were detected. (C) In vitro growth kinetics of the rTGEV viruses. ST cells were infected with the rTGEV-wt (wt, blue) and rTGEV-Δ7 (Δ7, red) viruses, at a moi of 5. Culture medium and total intracellular RNA were collected at different hours post infection. Intracellular RNA was only analyzed during those hours post infection in which viable cells were bound to the plate. Viral titers (left panel), and genomic RNA (gRNA) amounts (right panel), determined by RT-qPCR, were analyzed. Error bars represent the standard deviation from three independent experiments.
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ppat-1002090-g001: Generation of a recombinant TGEV virus lacking protein 7 expression (rTGEV-Δ7).(A) Genus α1 CoV protein 7a sequence alignment, using T-COFFEE [135]. Protein 7a sequences from the canine (CCoV) and porcine respiratory (PRCV) CoVs, transmissible gastroenteritis (TGEV) and feline infectious peritonitis (FIPV) viruses were used. GenBank accession numbers are ADB28914.1, ABG89313.1, CAA80842.1, and CAA62190.1, respectively. In silico prediction of TGEV protein 7 domains is represented. Transmembrane domains (TM) are in green [PredictProtein, [136]], the signal peptide in blue [Signal P3.0 Server, [137]], and a conserved phosphorilable Serine in red (S-Phos) [NetPhos 2.0 Server, [138]]. The predicted topology of TGEV protein 7 is also represented in lower panel [PSORTII [139]]. Signal peptide cleavage is indicated by a red arrowhead. S-Phos is indicated by a red star. (B) Mutations introduced to generate a rTGEV-Δ7 virus, right panel. The scheme of TGEV gRNA is shown in the upper part. The white letters represent the CS. Nucleotide change is indicated with a blue square, and the deletion (Δ) as a white square. Northern blot of subgenomic mRNAs (sgmRNAs) produced during rTGEV infections, right panel. ST cells were infected with rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) viruses, at a moi of 5. Total RNA was extracted at 8 hpi. The sgmRNAs for the spike (S), 3a, envelope (E), membrane (M), nucleocapsid (N) proteins, and protein 7 were detected. (C) In vitro growth kinetics of the rTGEV viruses. ST cells were infected with the rTGEV-wt (wt, blue) and rTGEV-Δ7 (Δ7, red) viruses, at a moi of 5. Culture medium and total intracellular RNA were collected at different hours post infection. Intracellular RNA was only analyzed during those hours post infection in which viable cells were bound to the plate. Viral titers (left panel), and genomic RNA (gRNA) amounts (right panel), determined by RT-qPCR, were analyzed. Error bars represent the standard deviation from three independent experiments.

Mentions: TGEV is a genus a1 CoV that contains three accessory genes: 3a, 3b and 7 [43], [44], [45]. The deletion of gene cluster 3ab demonstrated that these genes were not essential for in vitro and in vivo viral replication [45]. TGEV gene 7 is located at the 3′end of the genome, being the last ORF. In general, ORFs located in CoV genomes downstream of nucleocapsid (N) gene have been named as gene 7. And, one to three genes, 7a, 7b and 7c, have been described for several CoVs of genus α, β4 and γ3 at the end of their genomes [35], [46], [47] [48]. Nevertheless, most of these genes are not related to each other (J.L.G. Cruz, S. Zuñiga and L. Enjuanes, unpublished observations). In fact, new genes located in avian CoVs genomes after the N gene have been named differently as they showed no sequence homology to any other CoV genes [49]. TGEV protein 7 is similar to protein 7a of CoV genus α1, with a 72% homology to feline infectious peritonitis virus (FIPV), canine (CCoV) and porcine respiratory (PRCV) CoVs 7a proteins (Figure 1A) [50], [51]. The function of protein 7 has not been identified, and it has been proposed that it could play a role in virulence [52], [53]. The 7ab cluster deletion in FIPV (FIPV-Δ7ab) resulted in virus attenuation [54]. Nevertheless, the specific role that gene 7a plays in attenuation is not clear, as FIPV-Δ7ab phenotype was similar to the one observed for a FIPV isolate lacking only gene 7b [55].


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)

Generation of a recombinant TGEV virus lacking protein 7 expression (rTGEV-Δ7).(A) Genus α1 CoV protein 7a sequence alignment, using T-COFFEE [135]. Protein 7a sequences from the canine (CCoV) and porcine respiratory (PRCV) CoVs, transmissible gastroenteritis (TGEV) and feline infectious peritonitis (FIPV) viruses were used. GenBank accession numbers are ADB28914.1, ABG89313.1, CAA80842.1, and CAA62190.1, respectively. In silico prediction of TGEV protein 7 domains is represented. Transmembrane domains (TM) are in green [PredictProtein, [136]], the signal peptide in blue [Signal P3.0 Server, [137]], and a conserved phosphorilable Serine in red (S-Phos) [NetPhos 2.0 Server, [138]]. The predicted topology of TGEV protein 7 is also represented in lower panel [PSORTII [139]]. Signal peptide cleavage is indicated by a red arrowhead. S-Phos is indicated by a red star. (B) Mutations introduced to generate a rTGEV-Δ7 virus, right panel. The scheme of TGEV gRNA is shown in the upper part. The white letters represent the CS. Nucleotide change is indicated with a blue square, and the deletion (Δ) as a white square. Northern blot of subgenomic mRNAs (sgmRNAs) produced during rTGEV infections, right panel. ST cells were infected with rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) viruses, at a moi of 5. Total RNA was extracted at 8 hpi. The sgmRNAs for the spike (S), 3a, envelope (E), membrane (M), nucleocapsid (N) proteins, and protein 7 were detected. (C) In vitro growth kinetics of the rTGEV viruses. ST cells were infected with the rTGEV-wt (wt, blue) and rTGEV-Δ7 (Δ7, red) viruses, at a moi of 5. Culture medium and total intracellular RNA were collected at different hours post infection. Intracellular RNA was only analyzed during those hours post infection in which viable cells were bound to the plate. Viral titers (left panel), and genomic RNA (gRNA) amounts (right panel), determined by RT-qPCR, were analyzed. Error bars represent the standard deviation from three independent experiments.
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Related In: Results  -  Collection

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

ppat-1002090-g001: Generation of a recombinant TGEV virus lacking protein 7 expression (rTGEV-Δ7).(A) Genus α1 CoV protein 7a sequence alignment, using T-COFFEE [135]. Protein 7a sequences from the canine (CCoV) and porcine respiratory (PRCV) CoVs, transmissible gastroenteritis (TGEV) and feline infectious peritonitis (FIPV) viruses were used. GenBank accession numbers are ADB28914.1, ABG89313.1, CAA80842.1, and CAA62190.1, respectively. In silico prediction of TGEV protein 7 domains is represented. Transmembrane domains (TM) are in green [PredictProtein, [136]], the signal peptide in blue [Signal P3.0 Server, [137]], and a conserved phosphorilable Serine in red (S-Phos) [NetPhos 2.0 Server, [138]]. The predicted topology of TGEV protein 7 is also represented in lower panel [PSORTII [139]]. Signal peptide cleavage is indicated by a red arrowhead. S-Phos is indicated by a red star. (B) Mutations introduced to generate a rTGEV-Δ7 virus, right panel. The scheme of TGEV gRNA is shown in the upper part. The white letters represent the CS. Nucleotide change is indicated with a blue square, and the deletion (Δ) as a white square. Northern blot of subgenomic mRNAs (sgmRNAs) produced during rTGEV infections, right panel. ST cells were infected with rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) viruses, at a moi of 5. Total RNA was extracted at 8 hpi. The sgmRNAs for the spike (S), 3a, envelope (E), membrane (M), nucleocapsid (N) proteins, and protein 7 were detected. (C) In vitro growth kinetics of the rTGEV viruses. ST cells were infected with the rTGEV-wt (wt, blue) and rTGEV-Δ7 (Δ7, red) viruses, at a moi of 5. Culture medium and total intracellular RNA were collected at different hours post infection. Intracellular RNA was only analyzed during those hours post infection in which viable cells were bound to the plate. Viral titers (left panel), and genomic RNA (gRNA) amounts (right panel), determined by RT-qPCR, were analyzed. Error bars represent the standard deviation from three independent experiments.
Mentions: TGEV is a genus a1 CoV that contains three accessory genes: 3a, 3b and 7 [43], [44], [45]. The deletion of gene cluster 3ab demonstrated that these genes were not essential for in vitro and in vivo viral replication [45]. TGEV gene 7 is located at the 3′end of the genome, being the last ORF. In general, ORFs located in CoV genomes downstream of nucleocapsid (N) gene have been named as gene 7. And, one to three genes, 7a, 7b and 7c, have been described for several CoVs of genus α, β4 and γ3 at the end of their genomes [35], [46], [47] [48]. Nevertheless, most of these genes are not related to each other (J.L.G. Cruz, S. Zuñiga and L. Enjuanes, unpublished observations). In fact, new genes located in avian CoVs genomes after the N gene have been named differently as they showed no sequence homology to any other CoV genes [49]. TGEV protein 7 is similar to protein 7a of CoV genus α1, with a 72% homology to feline infectious peritonitis virus (FIPV), canine (CCoV) and porcine respiratory (PRCV) CoVs 7a proteins (Figure 1A) [50], [51]. The function of protein 7 has not been identified, and it has been proposed that it could play a role in virulence [52], [53]. The 7ab cluster deletion in FIPV (FIPV-Δ7ab) resulted in virus attenuation [54]. Nevertheless, the specific role that gene 7a plays in attenuation is not clear, as FIPV-Δ7ab phenotype was similar to the one observed for a FIPV isolate lacking only gene 7b [55].

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