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. 2015 Oct;96(10):3049-3058.
doi: 10.1099/jgv.0.000257. Epub 2015 Aug 6.

Porcine reproductive and respiratory syndrome virus 3C protease cleaves the mitochondrial antiviral signalling complex to antagonize IFN-β expression

Jianming Dong  1   2 , Shangen Xu  1   2 , Jing Wang  1   2 , Rui Luo  1   2 , Dang Wang  1   2 , Shaobo Xiao  1   2 , Liurong Fang  1   2 , Huanchun Chen  1   2 , Yunbo Jiang  2   1
Affiliations

Porcine reproductive and respiratory syndrome virus 3C protease cleaves the mitochondrial antiviral signalling complex to antagonize IFN-β expression

Jianming Dong et al. J Gen Virol. 2015 Oct.

Abstract

Porcine reproductive and respiratory syndrome, a highly infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), has developed various strategies to evade the host innate immune response, including the suppression of type I IFN activation. The mitochondrial antiviral signalling protein (MAVS) is an important bridging adaptor of retinoic acid-inducible gene I/melanoma differentiation-associated protein 5 signalling pathways. Here, we demonstrated that the 3C-like protease (3CLSP) of PRRSV prevented the induction of IFN-β by cleaving MAVS in a proteasome- and caspase-independent manner. Moreover, this cleavage ability was dependent on the protease activity of 3CLSP. Mutations specifically disrupting the cysteine protease activity of 3CLSP eliminated MAVS cleavage and the inhibition of IFN induction. Subsequently, we determined that 3CLSP cleaved MAVS at Glu268. Remarkably, a MAVS point mutation at Glu268 rendered MAVS resistant to 3CLSP cleavage. These results reveal a novel PRRSV mechanism to escape host immunity by directly cleaving MAVS.

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Figures

Fig. 1.
Fig. 1.
3CLSP inhibits the promoter activation of IFN-β, IRF3 and NF-κB, and the protease activity of 3CLSP is required to block SeV-mediated IFN-β induction. (a) PRRSV 3CLSP inhibits the promoter activation of IFN, IRF-3 and NF-κB. IFN-β–Luc, IRF3–Luc and NF-κB–Luc were co-transfected into HEK-293T cells, together with the pRL-TK plasmid and increasing quantities (0, 0.1, 0.2, 0.4 or 0.8 μg) of plasmids encoding 3CLSP. After 24 h transfection, the cells were treated with SeV for 8 h and then luciferase assays were performed. (b) 3CLSP blocks RIG-I-mediated IFN-β induction. HEK-293T cells were co-transfected with IFN-β–Luc, the pRL-TK plasmid and pCAGGS-HA-nsp4 together with constructs expressing RIG-I, its constitutively active mutant RIG-IN, MDA-5 or MAVS. Luciferase assays were performed 32 h after transfection. **P < 0.01 (3CLSP vs vector). Results are shown as means ± sd.
Fig. 2.
Fig. 2.
3CLSP suppression of IFN-β induction is dependent on protease activity. HEK-293T cells were co-transfected with IFN-β–Luc, IRF3–Luc or NF-κB–Luc along with pRL-TK plasmid and the 3CLSP mutants/3CLSP expression plasmids (0.8 μg). An empty vector (pCAGGS-HA) was used as a control. At 24 h post-transfection, cells were treated with SeV for 8 h and then luciferase activity was measured. *P < 0.05 (WT 3CLSP vs 3CLSP mutants). WT 3CLSP significantly inhibited IFN-β expression compared with the mutants and mock cells. Results are shown as means ± sd;ns, not significant.
Fig. 3.
Fig. 3.
PRRSV 3CLSP cleaves MAVS. (a) HEK-293T cells were transfected with Flag-tagged MAVS expression plasmids (3 μg) along with 3CLSP expression plasmids (0.5 μg). At 32 h after co-transfection, the cells were collected and analysed by Western blotting. (b) HEK-293T cells were transfected with Flag-tagged MAVS expression plasmids (3 μg) along with increasing quantities (0, 0.1, 0.2, 0.4 or 0.8 μg) of plasmid encoding 3CLSP. The cells were collected and analysed by Western blotting at 32 h post-transfection.
Fig. 4.
Fig. 4.
Marc-145 cells were infected with different doses (m.o.i. of 0.01, 0.1 and 1) of PRRSV or UV-PRRSV as a control. The cells were lysed 36 h p.i. and analysed by Western blotting (WB) using rabbit anti-MAVS antibody.
Fig. 5.
Fig. 5.
3CLSP activity is indispensable to MAVS cleavage. (a) HEK-293T cells were transfected with Flag-tagged MAVS expression plasmids (3 μg) along with the 3CLSP or 3CLSP mutant plasmids (0.5 μg). The cells were lysed at 32 h post-transfection and analysed by Western blotting (WB). (b) HEK-293T cells were transfected with Flag-tagged MAVS expression plasmids (3 μg) along with the 3CLSP expression plasmid or an empty vector (0.5 μg). At 24 h post-transfection, the cells were treated with MG132 (20 μM) or zVAD-FMK (20 μM) for 8 h. The cells were lysed and analysed by Western blotting.
Fig. 6.
Fig. 6.
3CLSP-mediated MAVS cleavage is involved in the inhibition of type I IFN induction. (a) Schematic representation of WT MAVS and its derivatives. HEK-293T cells were co-transfected with Flag-tagged WT MAVS or MAVS mutants (E268A, E274A and E288A) along with 3CLSP or an empty vector. These cells were lysed at 32 h post-transfection and analysed by Western blotting (WB). (b) HEK-293T cells were co-transfected with IFN-β–Luc, the pRL-TK plasmid and a plasmid encoding 3CLSP (0.3 μg) together with the empty vector, MAVS or MAVS (E268A) expression vector (0.3 μg). Luciferase activity was examined at 32 h post-transfection. The MAVS E268A mutant was significantly different from WT MAVS (*P < 0.01). (c) MAVS or MAVS truncated mutants are shown. Complete MAVS contained the N-terminal CARD domain, proline-rich domain and C-terminal TM domain. HEK-293T cells were co-transfected with the indicated reporter plasmid (0.1 μg), pRL-TK (0.1 μg) or the Flag-tagged MAVS expression plasmid (0.5 μg) along with a plasmid encoding Flag-tagged WT MAVS, 3CLSP-induced cleavage fragments of MAVS or an empty vector. **P < 0.01. Results are shown as means ± sd.
Fig. 6.
Fig. 6.
3CLSP-mediated MAVS cleavage is involved in the inhibition of type I IFN induction. (a) Schematic representation of WT MAVS and its derivatives. HEK-293T cells were co-transfected with Flag-tagged WT MAVS or MAVS mutants (E268A, E274A and E288A) along with 3CLSP or an empty vector. These cells were lysed at 32 h post-transfection and analysed by Western blotting (WB). (b) HEK-293T cells were co-transfected with IFN-β–Luc, the pRL-TK plasmid and a plasmid encoding 3CLSP (0.3 μg) together with the empty vector, MAVS or MAVS (E268A) expression vector (0.3 μg). Luciferase activity was examined at 32 h post-transfection. The MAVS E268A mutant was significantly different from WT MAVS (*P < 0.01). (c) MAVS or MAVS truncated mutants are shown. Complete MAVS contained the N-terminal CARD domain, proline-rich domain and C-terminal TM domain. HEK-293T cells were co-transfected with the indicated reporter plasmid (0.1 μg), pRL-TK (0.1 μg) or the Flag-tagged MAVS expression plasmid (0.5 μg) along with a plasmid encoding Flag-tagged WT MAVS, 3CLSP-induced cleavage fragments of MAVS or an empty vector. **P < 0.01. Results are shown as means ± sd.
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