Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence

Abstract

Viral immune evasion strategies target key aspects of the host antiviral response. Recently, it has been recognized that Toll-like receptors (TLRs) have a role in innate defense against viruses. Here, we define the function of the vaccinia virus (VV) protein A46R and show it inhibits intracellular signalling by a range of TLRs. TLR signalling is triggered by homotypic interactions between the Toll-like-interleukin-1 resistance (TIR) domains of the receptors and adaptor molecules. A46R contains a TIR domain and is the only viral TIR domain-containing protein identified to date. We demonstrate that A46R targets the host TIR adaptors myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like, TIR domain-containing adaptor inducing IFN-beta (TRIF), and the TRIF-related adaptor molecule and thereby interferes with downstream activation of mitogen-activated protein kinases and nuclear factor kappaB. TRIF mediates activation of interferon (IFN) regulatory factor 3 (IRF3) and induction of IFN-beta by TLR3 and TLR4 and suppresses VV replication in macrophages. Here, A46R disrupted TRIF-induced IRF3 activation and induction of the TRIF-dependent gene regulated on activation, normal T cell expressed and secreted. Furthermore, we show that A46R is functionally distinct from another described VV TLR inhibitor, A52R. Importantly, VV lacking the A46R gene was attenuated in a murine intranasal model, demonstrating the importance of A46R for VV virulence.

Figure 1.

A46R inhibits multiple IL-1–dependent signals. HEK 293 cells were transfected with 100 ng A46R or pcDNA3.1 (EV) and the NF-κB (a), p65 (b), JNK (c), or ERK (d) reporter plasmids as described in Materials and methods. 6 h before harvesting, the cells were stimulated with either 100 ng/ml IL-1 or 100 ng/ml TNF as indicated, and luciferase reporter gene activity was measured.

Figure 2.

A46R associates with MyD88 and blocks MyD88-dependent signaling. (a) Alignment of A46R with human TIR domains. The conserved motifs Box 1, Box 2, and Box 3 are indicated by a solid line. The eight differences between VV and variola virus A46R sequences are indicated by an asterisk. For A46R, amino acids 35–238 are shown. (b) HEK 293T cells were transfected with A46R and AU1-MyD88 (left) or Flag-TRAF2 (right) as indicated. After 24 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. (c) HEK 293T cells were transfected with 8 μg AU1-MyD88 (top) or Flag-TRAF2 (bottom). After 24 h, lysates were incubated with GST alone (lane 2) or GST-A46R (lane 3), and together with whole cell lysates (lane 1) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (d) HEK 293 cells were transfected with 4 μg of myc-MyD88. After 24 h, cells were infected with viruses either containing (vWT-A46R) or not (vΔA46R) the A46R gene (MOI = 1) and harvested 24 h after infection. Lysates were subjected to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. Whole cell lysates were analyzed for expression of A46R. (e) Murine macrophage RAW 264.7 cells were transfected with the phRL-TK reporter gene and the NF-κB luciferase construct as described in Materials and methods, together with pcDNA3.1 or 100 ng A46R. Cells were stimulated for 6 h with 10 nM MALP-2 (MALP), 5 μg/ml Pam3Cys (Pam), 1 μg/ml LPS, 250 ng/ml Flagellin (Flag), 1 μM R-848, or 5 μg/ml CpG DNA. Cells were harvested 24 h after transfection and the reporter gene activity was measured.

Figure 3.

A46R inhibits TLR4 signaling and interacts with TLR4, Mal, and TRAM. (a) HEK 293 cells were transfected with 50 ng CD4-TLR4, 25–100 ng A46R, or pcDNA3.1 (EV) and the NF-κB (left), p38 (middle), or ERK (right) reporter plasmids as indicated. Cells were harvested 24 h after transfection, and luciferase reporter gene activity was measured. (b–d) HEK 293T cells were transfected with A46R and Flag-TLR4 (b), Flag-Mal (c), or Flag-TRAM (d) as indicated. After 24 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. (e) HEK 293T cells were transfected with 8 μg of Flag-TLR4 (top), Flag-Mal (middle), or Flag-TRAM (bottom). After 24 h, lysates were incubated with GST-A46R (lane 3) or GST alone (lane 2), and together with whole cell lysate (lane 1), were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (f) HEK-TLR4 cells were transfected with the indicated amounts (ng) of A46R 24 h before stimulation with 1 μg/ml LPS, and 24 h after stimulation, supernatants were harvested and assayed for IL-8 by ELISA.

Figure 4.

A46R associates with TRIF and inhibits TRIF-dependent signaling and gene induction. (a) HEK 293T cells were transfected with A46R and Flag-TRIF as indicated. After 24 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. (b) HEK 293T cells were transfected with 8 μg Flag-TRIF. After 24 h, lysates were incubated with GST-A46R (lane 3) or GST alone (lane 2), and together with whole cell lysates (lane 1), were analyzed by SDS-PAGE and immunoblotting with anti-Flag Ab. (c) HEK 293 cells were transfected with the IRF3 reporter plasmids (as described in Materials and methods) with either 50 ng CD4-TLR4 (top) or 0.5 ng TLR3 (bottom) and 50–150 ng A46R or pcDNA3.1 (EV) as indicated. (bottom) Cells were stimulated with 25 μg/ml poly(I:C) 6 h before harvesting. Luciferase activity was measured after 24 h. (d) HEK-TLR3 cells were transfected with the indicated amounts (ng) of A46R 24 h before stimulation with 25 μg/ml poly(I:C), and 24 h after stimulation supernatants were harvested and assayed for RANTES by ELISA. (e and f) As in a and b, except Flag-SARM was transfected instead of Flag-TRIF.

Figure 5.

A46R is expressed early during virus infection and contributes to virus virulence. (a) BSC-1 cells were mock infected (M) or infected with VV Western reserve (MOI = 5) in the absence or presence of 40 μg/ml cytosine β-D-arabinofuranoside (AraC). Cells were harvested at the indicated times (h) after infection, and lysates were subjected to SDS-PAGE and immunoblotting, using either anti-A46R (top) or anti-D8L (bottom) Ab. (b) A46R contributes to virus virulence. Groups of 15 female, 6-wk-old Balb/c mice were infected intranasally with 5 × 10³ PFU of vWT-A46R, vΔA46R, or vA46R-REV. Each day, animals were weighed and the signs of illness were scored. Data are presented as the mean weight of each group of animals compared with the mean weight of the same group on day 0 (top graph), and the mean signs of illness score (bottom graph). Error bars are SEM. Asterisks represent days on which there was a statistically significant difference (P < 0.05; Student's t test) between the vWT-A46R and both control groups. (c) Number of cells recruited to the lungs of VV-infected mice. On days 2, 5, and 8 after infection, mice were killed, lungs were harvested, and cell suspensions were prepared. The total number of viable lung cells per animal was determined by Trypan blue exclusion. Data are means ± SEM.

Figure 6.

A46R and A52R target different TLR3-mediated signaling pathways. (a and b) HEK 293 cells were transfected with 0.5 ng TLR3 and the indicated amounts (ng) of either A52R or A46R, together with the NF-κB (a) or IRF3 (b) reporter plasmids as described in Materials and methods. Cells were stimulated with 25 μg/ml poly(I:C) 6 h before harvesting where indicated. Luciferase activity was measured after 24 h. Relative stimulation is shown on the y axis.