A46R and A52R from vaccinia virus are antagonists of host IL-1 and toll-like receptor signaling

Abstract

Poxviruses employ many strategies to evade and neutralize the host immune response. In this study, we have identified two vaccinia virus ORFs, termed A46R and A52R, that share amino acid sequence similarity with the Toll/IL-1 receptor (TIR) domain, a motif that defines the IL-1/Toll-like receptor (TLR) superfamily of receptors, which have a key role in innate immunity and inflammation. When expressed in mammalian cells, the protein products of both ORFs were shown to interfere specifically with IL-1 signal transduction. A46R partially inhibited IL-1-mediated activation of the transcription factor NFkappaB, and A52R potently blocked both IL-1- and TLR4-mediated NFkappaB activation. MyD88 is a TIR domain-containing adapter molecule known to have a central role in both IL-1 and TLR4 signaling. A52R mimicked the dominant-negative effect of a truncated version of MyD88 on IL-1, TLR4, and IL-18 signaling but had no effect on MyD88-independent signaling pathways. Therefore, A46R and A52R are likely to represent a mechanism used by vaccinia virus of suppressing TIR domain-dependent intracellular signaling.

Figure 1

Identification of A46R and A52R as potential members of the IL-1 receptor/TLR family. (A) Predicted amino acid sequence of A46R and A52R. The region of sequence similarity detected in a blast search with A46R is boxed. Identical amino acids are indicated by lines, and conservative substitutions are indicated by dots. The regions aligned with the family TIR domain in B are underlined. (B) Sequence comparison of the TIR domain of IL-1 receptor/TLR family members with A52R and A46R. For clarity, only those family members referred to in this article are shown. IL-1Rrp is the IL-18 receptor. Three conserved regions thought to be important in signaling are indicated by boxes.

Figure 2

Ectopic expression of epitope-tagged A46R and A52R in mammalian cells. 293 cells were transfected with equal amounts of DNA (12 μg) comprising empty vector (lanes 1 and 8); 3, 6, or 12 μg of A46R-Flag (lanes 2–4 and 9–11); or 3, 6, or 12 μg A52R-Flag (lanes 5–7 and 12–14). Proteins were then detected by immunoblotting either 24 h (lanes 1–7) or 48 h (lanes 8–14) later by using an anti-Flag antibody. The relevant molecular mass markers (in kDa) are shown on the right.

Figure 3

Effect of A46R and A52R on IL-1 signaling. 293 cells were transfected with 600 ng of empty vector (EV; black bars), 600 ng of vector encoding A46R (white bars), or 150 ng of vector encoding A52R (gray bars) for 48 h. At 6 h before harvesting, cells were stimulated with 100 ng/ml IL-1α or TNFα. NFκB reporter gene activity was then measured.

Figure 4

A52R and ΔMyD88 inhibit IL-1 signaling to NFκB. (A) Both A52R and ΔMyD88 inhibit the IL-1 but not the TNF pathway to NFκB. 293 cells were transfected with 300 ng of empty vector (EV; black bars), A52R (white bars), or ΔMyD88 (gray bars) for 24 h. At 6 h before harvesting, cells were stimulated with 100 ng/ml IL-1α or TNFα. NFκB reporter gene activity was then measured. (B) 293 cells were transfected with vectors encoding IL-1 signaling intermediates (150 ng each of IL-1RI and IL-1RAcP, 300 ng of IL-1RAcP or MyD88, or 150 ng of IRAK) together with 300 ng (or 450 ng for IRAK) of a vector encoding either A52R (Upper) or ΔMyD88 (Lower) for 24 h. NFκB reporter gene activity was then measured. (C) A52R inhibits IL-1-induced IL-8 promoter activation but not GH-induced lactogenesis hormone response element activation. (Left) HeLa cells were transfected with 10 ng of IL-1R1 and IL-1RAcP or MyD88 in the presence of 100 ng of empty vector (black bars) or vector encoding A52R (white bars). After 24–30 h, IL-8 promoter activity was measured by a reporter gene assay. (Right) HeLa cells were transfected with 100 ng of empty vector (black bar) or vector encoding A52R (white bar) 18 h before stimulation with 1 ng/ml growth hormone (GH) for 6 h. Lactogenesis hormone response element activity was measured by a reporter gene assay. Data are expressed as stimulation by GH over control in the absence or presence of A52R.

Figure 5

Inhibition of TLR4 and IL-18 signaling by A52R and ΔMyD88. (A) 293 cells were cotransfected with 150 ng of a vector encoding TLR4 together with 450 ng of empty vector (EV; black bars), A52R (white bars), or ΔMyD88 (gray bars) for 24 h. NFκB reporter gene activity was then measured. (B) 293 cells were cotransfected with 300 ng of a vector encoding AcPL together with 300 ng of empty vector (black bars), A52R (white bars), or ΔMyD88 (gray bars) for 48 h. At 6 h before harvesting, cells were stimulated with 20 ng/ml IL-18. NFκB reporter gene activity was then measured.