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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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Cellular RNA integrity.(A) Total RNA extracted from infected ST cells, at indicated times post infection, was analyzed using a Bioanalyzer. The position of 28S and 18S rRNAs are indicated. (B) 28S rRNA integrity. Graph of 28S fluorescence intensity, as measured by Bioanalyzer, in the RNA samples from ST cells infected with rTGEV-wt (blue) or rTGEV-Δ7 (red), collected at different times post infection. f.u., fluorescence units. Error bars indicate the standard deviation from three independent experiments. *, p-value <0.05. (C) ST cells were treated with caspase inhibitor ZVAD, and infected. Total RNA was extracted and analyzed using a Bioanalyzer. (D) ST cells were transfected with Poly(I:C), and total RNA was extracted 16 hours post transfection. ST cells were also infected with a vaccinia virus expressing T7 polymerase (T7), or with the vaccinia expressing T7 polymerase, and two additional vaccinia viruses expressing 2′-5′ OAS and RNase L (RL+OAS). Total RNA was extracted 24 hpi. In all cases, cell RNA integrity was analyzed using a Bioanalyzer.
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ppat-1002090-g005: Cellular RNA integrity.(A) Total RNA extracted from infected ST cells, at indicated times post infection, was analyzed using a Bioanalyzer. The position of 28S and 18S rRNAs are indicated. (B) 28S rRNA integrity. Graph of 28S fluorescence intensity, as measured by Bioanalyzer, in the RNA samples from ST cells infected with rTGEV-wt (blue) or rTGEV-Δ7 (red), collected at different times post infection. f.u., fluorescence units. Error bars indicate the standard deviation from three independent experiments. *, p-value <0.05. (C) ST cells were treated with caspase inhibitor ZVAD, and infected. Total RNA was extracted and analyzed using a Bioanalyzer. (D) ST cells were transfected with Poly(I:C), and total RNA was extracted 16 hours post transfection. ST cells were also infected with a vaccinia virus expressing T7 polymerase (T7), or with the vaccinia expressing T7 polymerase, and two additional vaccinia viruses expressing 2′-5′ OAS and RNase L (RL+OAS). Total RNA was extracted 24 hpi. In all cases, cell RNA integrity was analyzed using a Bioanalyzer.

Mentions: In principle, RNA decay could be responsible for the observed translational shutoff. Therefore, total cellular RNA integrity was evaluated using a Bioanalyzer [75], [76], [77]. Wild-type virus infection induced a modest RNA processing, especially at 24 hpi (Figure 5A). In contrast, rTGEV-Δ7 infection induced a faster and stronger cellular RNA degradation (Figure 5A). This data indicated that the cellular translational shutoff could be due, at least in part, to cellular mRNA degradation. Moreover, the increase in 28S rRNA degradation (Figure 5B), could affect both cellular and viral protein synthesis [78]. Nucleases activated by cell apoptosis could be responsible for the observed RNA degradation [79]. To study whether this was the case, we took advantage of the previous description of the inhibition of TGEV induced apoptosis by the addition of caspases inhibitor ZVAD, without affecting virus production [63]. In fact, after infection of ST cells with wt or rTGEV-Δ7 viruses in the presence of ZVAD, no CPE was observed. Total RNA was extracted from non-treated or ZVAD-treated cells, and the same RNA degradation patterns were observed in both cases (Figure 5C), indicating that the increased RNA degradation caused by rTGEV-Δ7 virus was independent of nucleases activated by cell apoptosis. To determine whether the observed cellular RNA cleavage was due to a dsRNA induced antiviral response, ST cells were treated with polyinosinic-polycytidylic acid [Poly(I:C)], which is a potent activator of this type of response [77], [80], [81]. Cells transfected with Poly(I:C) showed the same RNA degradation pattern as those infected with the rTGEV-Δ7 and parental viruses (Figure 5D), in contrast to mock treated cells. These results suggested that the cellular RNA cleavage increase, during rTGEV-Δ7 infection, was due to an enhancement of dsRNA induced antiviral activity. In general, the main effector of this process is RNase L [81], [82], [83]. To further analyze the relevance of this nuclease during TGEV infection, a recombinant vaccinia virus (VV) system was used. It was previously described that VV does not induce strong RNA degradation, due to the presence of viral genes that inhibit the RNase L system. To efficiently trigger dsRNA activated RNA degradation by RNase L, cells must be infected by VV expressing 2′-5′ OAS and RNase L [84]. Taking advantage of the wide host range of VV, porcine ST cells were infected with VV, or VVs expressing 2′-5′ OAS and RNase L. As expected, VV induced a very slight RNA degradation, that was increased by the co-expression of 2′-5′ OAS and RNase L (Figure 5D). Moreover, the RNA degradation pattern produced by the expression of RNase L system was identical to the one observed after rTGEV-Δ7 infection, strongly suggesting that RNaseL is the main nuclease involved in the increased RNA degradation after rTGEV-Δ7 infection.


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)

Cellular RNA integrity.(A) Total RNA extracted from infected ST cells, at indicated times post infection, was analyzed using a Bioanalyzer. The position of 28S and 18S rRNAs are indicated. (B) 28S rRNA integrity. Graph of 28S fluorescence intensity, as measured by Bioanalyzer, in the RNA samples from ST cells infected with rTGEV-wt (blue) or rTGEV-Δ7 (red), collected at different times post infection. f.u., fluorescence units. Error bars indicate the standard deviation from three independent experiments. *, p-value <0.05. (C) ST cells were treated with caspase inhibitor ZVAD, and infected. Total RNA was extracted and analyzed using a Bioanalyzer. (D) ST cells were transfected with Poly(I:C), and total RNA was extracted 16 hours post transfection. ST cells were also infected with a vaccinia virus expressing T7 polymerase (T7), or with the vaccinia expressing T7 polymerase, and two additional vaccinia viruses expressing 2′-5′ OAS and RNase L (RL+OAS). Total RNA was extracted 24 hpi. In all cases, cell RNA integrity was analyzed using a Bioanalyzer.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002090-g005: Cellular RNA integrity.(A) Total RNA extracted from infected ST cells, at indicated times post infection, was analyzed using a Bioanalyzer. The position of 28S and 18S rRNAs are indicated. (B) 28S rRNA integrity. Graph of 28S fluorescence intensity, as measured by Bioanalyzer, in the RNA samples from ST cells infected with rTGEV-wt (blue) or rTGEV-Δ7 (red), collected at different times post infection. f.u., fluorescence units. Error bars indicate the standard deviation from three independent experiments. *, p-value <0.05. (C) ST cells were treated with caspase inhibitor ZVAD, and infected. Total RNA was extracted and analyzed using a Bioanalyzer. (D) ST cells were transfected with Poly(I:C), and total RNA was extracted 16 hours post transfection. ST cells were also infected with a vaccinia virus expressing T7 polymerase (T7), or with the vaccinia expressing T7 polymerase, and two additional vaccinia viruses expressing 2′-5′ OAS and RNase L (RL+OAS). Total RNA was extracted 24 hpi. In all cases, cell RNA integrity was analyzed using a Bioanalyzer.
Mentions: In principle, RNA decay could be responsible for the observed translational shutoff. Therefore, total cellular RNA integrity was evaluated using a Bioanalyzer [75], [76], [77]. Wild-type virus infection induced a modest RNA processing, especially at 24 hpi (Figure 5A). In contrast, rTGEV-Δ7 infection induced a faster and stronger cellular RNA degradation (Figure 5A). This data indicated that the cellular translational shutoff could be due, at least in part, to cellular mRNA degradation. Moreover, the increase in 28S rRNA degradation (Figure 5B), could affect both cellular and viral protein synthesis [78]. Nucleases activated by cell apoptosis could be responsible for the observed RNA degradation [79]. To study whether this was the case, we took advantage of the previous description of the inhibition of TGEV induced apoptosis by the addition of caspases inhibitor ZVAD, without affecting virus production [63]. In fact, after infection of ST cells with wt or rTGEV-Δ7 viruses in the presence of ZVAD, no CPE was observed. Total RNA was extracted from non-treated or ZVAD-treated cells, and the same RNA degradation patterns were observed in both cases (Figure 5C), indicating that the increased RNA degradation caused by rTGEV-Δ7 virus was independent of nucleases activated by cell apoptosis. To determine whether the observed cellular RNA cleavage was due to a dsRNA induced antiviral response, ST cells were treated with polyinosinic-polycytidylic acid [Poly(I:C)], which is a potent activator of this type of response [77], [80], [81]. Cells transfected with Poly(I:C) showed the same RNA degradation pattern as those infected with the rTGEV-Δ7 and parental viruses (Figure 5D), in contrast to mock treated cells. These results suggested that the cellular RNA cleavage increase, during rTGEV-Δ7 infection, was due to an enhancement of dsRNA induced antiviral activity. In general, the main effector of this process is RNase L [81], [82], [83]. To further analyze the relevance of this nuclease during TGEV infection, a recombinant vaccinia virus (VV) system was used. It was previously described that VV does not induce strong RNA degradation, due to the presence of viral genes that inhibit the RNase L system. To efficiently trigger dsRNA activated RNA degradation by RNase L, cells must be infected by VV expressing 2′-5′ OAS and RNase L [84]. Taking advantage of the wide host range of VV, porcine ST cells were infected with VV, or VVs expressing 2′-5′ OAS and RNase L. As expected, VV induced a very slight RNA degradation, that was increased by the co-expression of 2′-5′ OAS and RNase L (Figure 5D). Moreover, the RNA degradation pattern produced by the expression of RNase L system was identical to the one observed after rTGEV-Δ7 infection, strongly suggesting that RNaseL is the main nuclease involved in the increased RNA degradation after rTGEV-Δ7 infection.

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