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A novel TLR3 inhibitor encoded by African swine fever virus (ASFV).

de Oliveira VL, Almeida SC, Soares HR, Crespo A, Marshall-Clarke S, Parkhouse RM - Arch. Virol. (2011)

Bottom Line: Consistent with this, expression of I329L protein also inhibited the activation of interferon-β and CCL5.Finally, overexpression of TRIF reversed I329L-mediated inhibition of both NFκB and IRF3 activation.Our results suggest that TRIF, a key MyD88-independent adaptor molecule, is a possible target of this viral host modulation gene.

View Article: PubMed Central - PubMed

Affiliation: Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, Oeiras, Portugal.

ABSTRACT
African swine fever virus (ASFV) encodes proteins that manipulate important host antiviral mechanisms. Bioinformatic analysis of the ASFV genome revealed ORF I329L, a gene without any previous functional characterization as a possible inhibitor of TLR signaling. We demonstrate that ORF I329L encodes a highly glycosylated protein expressed in the cell membrane and on its surface. I329L also inhibited dsRNA-stimulated activation of NFκB and IRF3, two key players in innate immunity. Consistent with this, expression of I329L protein also inhibited the activation of interferon-β and CCL5. Finally, overexpression of TRIF reversed I329L-mediated inhibition of both NFκB and IRF3 activation. Our results suggest that TRIF, a key MyD88-independent adaptor molecule, is a possible target of this viral host modulation gene. The demonstration of an ASFV host evasion molecule inhibiting TLR responses is consistent with the ability of this virus to infect vertebrate and invertebrate hosts, both of which deploy innate immunity controlled by conserved TLR systems.

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I329L inhibits activation of the IFN-β promoter at the level of TRIF. 6A) I329L inhibits TRIF-mediated activation of IFN-β. Activation of the IFN-β promoter reporter plasmid was induced via ectopic expression of TRIF in HEK-293T cells through transfection with the TRIF plasmid vector. Transfections were performed with equal amounts of DNA comprising 25 ng of TRIF plasmid vector (TRIF) in the presence of increasing amounts of I329L plasmid vector (100–300 ng) (pcDNA3-I329L) jointly with IFN-β-reporter plasmids (IFN-β). 6B) Overexpression of TRIF reverses I329L-mediated inhibition of IFN-β activation. TRIF was ectopically expressed in HEK-293T cells with increasing amounts of TRIF plasmid vector (25–100 ng) (TRIF) in the presence of 200 ng of the plasmid vector coding for I329L (pcDNA3-I329L) simultaneously with 100 ng of IFN-β-reporter plasmid vector (IFN-β) plus 25 ng of β-gal plasmid vector (β-gal) and after stimulating for 6 hours with 25 μg/ml of poly (I:C) where indicated, before harvesting the cells. In all cases, luciferase activity was measured after 48 h. Luciferase activity was normalized to the β-galactosidase activity obtained with the cotransfected β-gal plasmid internal control. Standard deviations are shown by error bars. Details are in Materials and methods
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Fig6: I329L inhibits activation of the IFN-β promoter at the level of TRIF. 6A) I329L inhibits TRIF-mediated activation of IFN-β. Activation of the IFN-β promoter reporter plasmid was induced via ectopic expression of TRIF in HEK-293T cells through transfection with the TRIF plasmid vector. Transfections were performed with equal amounts of DNA comprising 25 ng of TRIF plasmid vector (TRIF) in the presence of increasing amounts of I329L plasmid vector (100–300 ng) (pcDNA3-I329L) jointly with IFN-β-reporter plasmids (IFN-β). 6B) Overexpression of TRIF reverses I329L-mediated inhibition of IFN-β activation. TRIF was ectopically expressed in HEK-293T cells with increasing amounts of TRIF plasmid vector (25–100 ng) (TRIF) in the presence of 200 ng of the plasmid vector coding for I329L (pcDNA3-I329L) simultaneously with 100 ng of IFN-β-reporter plasmid vector (IFN-β) plus 25 ng of β-gal plasmid vector (β-gal) and after stimulating for 6 hours with 25 μg/ml of poly (I:C) where indicated, before harvesting the cells. In all cases, luciferase activity was measured after 48 h. Luciferase activity was normalized to the β-galactosidase activity obtained with the cotransfected β-gal plasmid internal control. Standard deviations are shown by error bars. Details are in Materials and methods

Mentions: A direct effect of I329L on TRIF signaling was demonstrated using HEK-TLR3 cells transfected with TRIF as, in the presence of I329L, the IFN-β signaling pathway was inhibited (Fig. 6A). To further investigate the possible impact of I329L on TRIF signaling, HEK-TLR3 cells were simultaneously transfected with I329L (pcDNA3-I329L), the IFN-β luciferase reporter plasmid (IFN-β) and increasing quantities of TRIF plasmid (TRIF) and then stimulated with poly (I:C). As can be seen, overexpression of TRIF reversed the inhibition of reporter activation induced by I329L in a dose-dependent manner, consistent with the hypothesis that the ASFV gene I329L targets TRIF signaling (Fig. 6B).Fig. 6


A novel TLR3 inhibitor encoded by African swine fever virus (ASFV).

de Oliveira VL, Almeida SC, Soares HR, Crespo A, Marshall-Clarke S, Parkhouse RM - Arch. Virol. (2011)

I329L inhibits activation of the IFN-β promoter at the level of TRIF. 6A) I329L inhibits TRIF-mediated activation of IFN-β. Activation of the IFN-β promoter reporter plasmid was induced via ectopic expression of TRIF in HEK-293T cells through transfection with the TRIF plasmid vector. Transfections were performed with equal amounts of DNA comprising 25 ng of TRIF plasmid vector (TRIF) in the presence of increasing amounts of I329L plasmid vector (100–300 ng) (pcDNA3-I329L) jointly with IFN-β-reporter plasmids (IFN-β). 6B) Overexpression of TRIF reverses I329L-mediated inhibition of IFN-β activation. TRIF was ectopically expressed in HEK-293T cells with increasing amounts of TRIF plasmid vector (25–100 ng) (TRIF) in the presence of 200 ng of the plasmid vector coding for I329L (pcDNA3-I329L) simultaneously with 100 ng of IFN-β-reporter plasmid vector (IFN-β) plus 25 ng of β-gal plasmid vector (β-gal) and after stimulating for 6 hours with 25 μg/ml of poly (I:C) where indicated, before harvesting the cells. In all cases, luciferase activity was measured after 48 h. Luciferase activity was normalized to the β-galactosidase activity obtained with the cotransfected β-gal plasmid internal control. Standard deviations are shown by error bars. Details are in Materials and methods
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Fig6: I329L inhibits activation of the IFN-β promoter at the level of TRIF. 6A) I329L inhibits TRIF-mediated activation of IFN-β. Activation of the IFN-β promoter reporter plasmid was induced via ectopic expression of TRIF in HEK-293T cells through transfection with the TRIF plasmid vector. Transfections were performed with equal amounts of DNA comprising 25 ng of TRIF plasmid vector (TRIF) in the presence of increasing amounts of I329L plasmid vector (100–300 ng) (pcDNA3-I329L) jointly with IFN-β-reporter plasmids (IFN-β). 6B) Overexpression of TRIF reverses I329L-mediated inhibition of IFN-β activation. TRIF was ectopically expressed in HEK-293T cells with increasing amounts of TRIF plasmid vector (25–100 ng) (TRIF) in the presence of 200 ng of the plasmid vector coding for I329L (pcDNA3-I329L) simultaneously with 100 ng of IFN-β-reporter plasmid vector (IFN-β) plus 25 ng of β-gal plasmid vector (β-gal) and after stimulating for 6 hours with 25 μg/ml of poly (I:C) where indicated, before harvesting the cells. In all cases, luciferase activity was measured after 48 h. Luciferase activity was normalized to the β-galactosidase activity obtained with the cotransfected β-gal plasmid internal control. Standard deviations are shown by error bars. Details are in Materials and methods
Mentions: A direct effect of I329L on TRIF signaling was demonstrated using HEK-TLR3 cells transfected with TRIF as, in the presence of I329L, the IFN-β signaling pathway was inhibited (Fig. 6A). To further investigate the possible impact of I329L on TRIF signaling, HEK-TLR3 cells were simultaneously transfected with I329L (pcDNA3-I329L), the IFN-β luciferase reporter plasmid (IFN-β) and increasing quantities of TRIF plasmid (TRIF) and then stimulated with poly (I:C). As can be seen, overexpression of TRIF reversed the inhibition of reporter activation induced by I329L in a dose-dependent manner, consistent with the hypothesis that the ASFV gene I329L targets TRIF signaling (Fig. 6B).Fig. 6

Bottom Line: Consistent with this, expression of I329L protein also inhibited the activation of interferon-β and CCL5.Finally, overexpression of TRIF reversed I329L-mediated inhibition of both NFκB and IRF3 activation.Our results suggest that TRIF, a key MyD88-independent adaptor molecule, is a possible target of this viral host modulation gene.

View Article: PubMed Central - PubMed

Affiliation: Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, Oeiras, Portugal.

ABSTRACT
African swine fever virus (ASFV) encodes proteins that manipulate important host antiviral mechanisms. Bioinformatic analysis of the ASFV genome revealed ORF I329L, a gene without any previous functional characterization as a possible inhibitor of TLR signaling. We demonstrate that ORF I329L encodes a highly glycosylated protein expressed in the cell membrane and on its surface. I329L also inhibited dsRNA-stimulated activation of NFκB and IRF3, two key players in innate immunity. Consistent with this, expression of I329L protein also inhibited the activation of interferon-β and CCL5. Finally, overexpression of TRIF reversed I329L-mediated inhibition of both NFκB and IRF3 activation. Our results suggest that TRIF, a key MyD88-independent adaptor molecule, is a possible target of this viral host modulation gene. The demonstration of an ASFV host evasion molecule inhibiting TLR responses is consistent with the ability of this virus to infect vertebrate and invertebrate hosts, both of which deploy innate immunity controlled by conserved TLR systems.

Show MeSH
Related in: MedlinePlus