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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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Homology with TLR3 in the intracellular domain of ASFV protein I329L. The analysis presented here was performed with ClustalW2. 1A) Sequence of the intracellular domain of I329L (amino acid residues 260 to 329) indicating the predicted BOX1 and BOX2 regions. 1B) Predicted TIR-like domain in the BOX1 domain of I329L has sequence homology with known proteins containing the BOX1 TIR domain. 1C) Predicted TIR-like domain in the BOX 2 domain of I329L. Details are in Materials and methods
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Fig1: Homology with TLR3 in the intracellular domain of ASFV protein I329L. The analysis presented here was performed with ClustalW2. 1A) Sequence of the intracellular domain of I329L (amino acid residues 260 to 329) indicating the predicted BOX1 and BOX2 regions. 1B) Predicted TIR-like domain in the BOX1 domain of I329L has sequence homology with known proteins containing the BOX1 TIR domain. 1C) Predicted TIR-like domain in the BOX 2 domain of I329L. Details are in Materials and methods

Mentions: Bioinformatic analysis of the sequence of pI329L from the tissue-culture-adapted non-pathogenic ASFV isolate Ba71V (Uniprot database code I329_ASFB7), which grows in a variety of commonly used fibroblast cell lines, reveals a type I membrane structure with a 17-amino-acid signal peptide (amino acids 1-17), followed by an N-terminal extracellular domain (amino acids 18-239), a transmembrane domain (amino acids 240-260), and a 69-amino-acid C-terminal intracellular domain (amino acids 261-329). A number of potential glycosylation sites were indicated, at amino acids 32, 39, 44, 76, 82, 101, 185, and 219. Simple sequence examination of the sequence database did not show any gene similar to I329L; however, after multiple alignments with several TLR proteins, a weak but interesting alignment between I329L and TLR from Drosophila melanogaster was revealed. Importantly, the intracellular domain of I329L aligned with BOX1 and BOX2 regions of the human TIR-like domain of TLR3 (Fig. 1A), with a sequence similarity of 35% to TLR3 in BOX1 (Fig. 1B). Furthermore, the BOX2 of TLR3 also showed similarities with the I329L intracellular domain (Fig. 1C). Finally, the conserved extracellular region with leucine-rich repeats (LRRs) is an important motif for interaction between proteins and is common to several TLRs. Although the observed similarity was admittedly weak, we nonetheless decided to clone and test I329L for its impact on poly (I:C)-stimulated innate immunity in reporter assays (see below).Fig. 1


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)

Homology with TLR3 in the intracellular domain of ASFV protein I329L. The analysis presented here was performed with ClustalW2. 1A) Sequence of the intracellular domain of I329L (amino acid residues 260 to 329) indicating the predicted BOX1 and BOX2 regions. 1B) Predicted TIR-like domain in the BOX1 domain of I329L has sequence homology with known proteins containing the BOX1 TIR domain. 1C) Predicted TIR-like domain in the BOX 2 domain of I329L. Details are in Materials and methods
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Related In: Results  -  Collection

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Fig1: Homology with TLR3 in the intracellular domain of ASFV protein I329L. The analysis presented here was performed with ClustalW2. 1A) Sequence of the intracellular domain of I329L (amino acid residues 260 to 329) indicating the predicted BOX1 and BOX2 regions. 1B) Predicted TIR-like domain in the BOX1 domain of I329L has sequence homology with known proteins containing the BOX1 TIR domain. 1C) Predicted TIR-like domain in the BOX 2 domain of I329L. Details are in Materials and methods
Mentions: Bioinformatic analysis of the sequence of pI329L from the tissue-culture-adapted non-pathogenic ASFV isolate Ba71V (Uniprot database code I329_ASFB7), which grows in a variety of commonly used fibroblast cell lines, reveals a type I membrane structure with a 17-amino-acid signal peptide (amino acids 1-17), followed by an N-terminal extracellular domain (amino acids 18-239), a transmembrane domain (amino acids 240-260), and a 69-amino-acid C-terminal intracellular domain (amino acids 261-329). A number of potential glycosylation sites were indicated, at amino acids 32, 39, 44, 76, 82, 101, 185, and 219. Simple sequence examination of the sequence database did not show any gene similar to I329L; however, after multiple alignments with several TLR proteins, a weak but interesting alignment between I329L and TLR from Drosophila melanogaster was revealed. Importantly, the intracellular domain of I329L aligned with BOX1 and BOX2 regions of the human TIR-like domain of TLR3 (Fig. 1A), with a sequence similarity of 35% to TLR3 in BOX1 (Fig. 1B). Furthermore, the BOX2 of TLR3 also showed similarities with the I329L intracellular domain (Fig. 1C). Finally, the conserved extracellular region with leucine-rich repeats (LRRs) is an important motif for interaction between proteins and is common to several TLRs. Although the observed similarity was admittedly weak, we nonetheless decided to clone and test I329L for its impact on poly (I:C)-stimulated innate immunity in reporter assays (see below).Fig. 1

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