Peste des petits ruminants virus non-structural C protein inhibits the induction of interferon-β by potentially interacting with MAVS and RIG-I

Abstract

Peste des petits ruminants virus (PPRV) causes an acute and highly contagious disease in domestic and wild small ruminants throughout the world, mainly by invoking immunosuppression in its natural hosts. It has been suggested that the non-structural C protein of PPRV helps in evading host responses but the molecular mechanisms by which it antagonizes the host responses have not been fully characterized. Here, we report the antagonistic effect of PPRV C protein on the expression of interferon-β (IFN-β) through both MAVS and RIG-I mediated pathways in vitro. Dual luciferase reporter assay and direct expression of IFN-β mRNA analysis indicated that PPRV C significantly down regulates IFN-β via its potential interaction with MAVS and RIG-I signaling molecules. Results further indicated that PPRV C protein significantly suppresses endogenous and exogenous IFN-β-induced anti-viral effects in PPRV, EMCV and SVS infections in vitro. Moreover, PPRV C protein not only down regulates IFN-β but also the downstream cytokines of interferon stimulated genes 56 (ISG56), ISG15, C-X-C motif chemokine (CXCL10) and RIG-I mediated activation of IFN promoter elements of ISRE and NF-κB. Further, this study deciphers that PPRV C protein could significantly inhibit the phosphorylation of STAT1 and interferes with the signal transmission in JAK-STAT signaling pathway. Collectively, this study indicates that PPRV C protein is important for innate immune evasion and disease progression.

Keywords: Antiviral state; Interferon-β (IFN-β); Luciferase reporter assay; Pest des petits ruminants virus; Protein C.

Fig. 1

PPRV C protein inhibits the gene expression of INF-β and its downstream factors induced by RIG-IN at transcriptional level. HEK-293T cells (2 × 10⁵) in 6-well plates were cultured (70–80% confluency) and were transfected with pCMV-HA (2.5 µg), pCMV-HA-C (2.5 µg), pRK-Flag-RIG-IN (1 µg) alone, and co-transfected with pCMV-HA-C (1.5 µg) and pRK-Flag-RIG-IN (1 µg) using Lipofectamine 2000 Transfection Reagent. At 24 h post transfection (hpt), the expression of Flag-RIG-IN and HA-C was detected by western blotting with anti-Flag mAb and anti-HA mAb, respectively. The expression of β-actin was used as a loading control. The mRNA expression of each IFN-β, ISG56, ISG15 and CXCL-10 was measured by RT-qPCR. a Western blotting has shown that HA-C and Flag-RIG-IN expressed in the transfected cells. b The mRNA expression of IFN-β has been reduced to almost half under the effect of PPRV C as compared to RIG-1N. Similarly, mRNA expression of ISG56 (c) ISG15 (d) and CXCL-10 (e) have been down-regulated by PPRV C expression

Fig. 2

PPRV C protein inhibits RIG-IN mediated reporter gene activity. HEK-293T cells (1 × 10⁵) in 12-well plates were cultured (70–80% confluency) and were transfected with internal plasmid pRL-TK (10 ng) along with reporter plasmids pGL3-IFN-β (100 ng) (a), pGL3-ISRE (100 ng) (b) and pGL3-NF-κB (100 ng) (c) in each well, respectively. Then cells were transfected with pCMV-HA (500 ng), pCMV-HA-C (400 ng) and pRK-Flag-RIG-IN (100 ng) alone, and with pCMV-HA-C (400 ng) and pRK-Flag-RIG-IN (100 ng) together using Lipofectamine 2000 Transfection Reagent. Empty vectors were added to ensure cells received equal amount of plasmids in each transfection. At 24 hpt, cell lysates were analyzed for Firefly and Renilla luciferase activity using a dual-luciferase reporter assay kits. The expression levels of IFN-β (a), ISRE (b) and NF-κB (c) were significantly reduced in pCMV-HA-C and pRK-Flag-RIG-IN co-transfected cells than that of pRK-Flag-RIG-IN transfected cells

Fig. 3

Effect of the expression of the seven possible target molecules of PPRV C protein-induced activation of IFN-β promoter in RLR pathway. Monolayer HEK-293T cells in 12-well plates were transfected with internal reference gene plasmid pRL-TK (10 ng) and reporter gene plasmid pGL3-IFN-β (100 ng) in each well. Cells were then transfected with pRK-Flag-RIG-IN (100 ng), pRK-Flag-MAVS (100 ng), pRK-Flag-IKKε (100 ng), pRK-Flag-TBKI (100 ng), pRK-Flag-TRAF3 (100 ng), pRK-Flag-IRF3 (100 ng), and pRK-Flag-IRF7 (100 ng) alone and together with pCMV-HA-C (400 ng) in each well. For in depth analysis, various doses of pCMV-HA-C (100, 250 or 400 ng) against constant values of either pRK-Flag-RIG-IN (100 ng) or pRK-Flag-MAVS (100 ng) were used (h and i). Empty vectors were added to ensure cells received equal amount of plasmids in each transfection. At 24 hpt, cell lysates were analyzed for Firefly and Renilla luciferase activity using a dual-luciferase reporter assay kit. Expression of IFN-β responsive promoter under the effect of C protein was significantly lower than activated by RIG-I (a) and by MAVS (b). On the contrary, the effect of C protein on the expression levels of IKKε (c), TBKI (d), TRAF3 (e), IRF3 (f), and IRF7 (g) was not significant. h RIG-I-mediated IFN-β and MAVS-mediated IFN-β promoter activation were found to be significantly inhibited by C protein (h and i respectively) and were dose dependent; increase in dose from 100 to 400 ng halted IFN-β expression in each case

Fig. 4

PPRV C protein inhibits the anti-viral effect via decreasing the production of endogenous IFN response. HEK-293T cells (2 × 10⁵) in 6-well culture plates were cultured in DMEM supplemented with 10% FBS for growing to 70–80% confluency. Cells were transfected with pCMV-HA (2.5 µg), pCMV-HA-C (2.5 µg) and pRK-Flag-RIG-IN (1 µg) alone, and pCMV-HA-C (1.5 µg) and pRK-Flag-RIG-IN (1 µg) together using Lipofectamine 2000 Transfection Reagent according to the manufacturer’s instructions. Empty vectors were added to ensure cells received equal amount of plasmids in each transfection. At 24 hpt, the supernatants were harvested and then 1 ml supernatants and 1 ml fresh DMEM medium were added to the new HEK-293T cells and cultured for 24 h, followed by infection with 1 MOI PPRV, 0.1 MOI VSV and 0.1 MOI EMCV, respectively. At 24 h post infection (hpi), all infected cells were harvested for RT-qPCR to measure the gene expression of PPRV H, EMCV VP1 and VSV L. a The expression of PPRV H gene in RIG-IN and PPRV C co-transfected cells was most significantly higher than that in RIG-IN transfected cells (p < 0.001); b The expression of EMCV VP1 gene in RIG-IN and PPRV C co-transfected cells was most significantly higher than that in RIG-IN transfected cells (p < 0.001); c The expression of VSV L gene in RIG-IN and PPRV C co-transfected cells was most significantly higher than that in RIG-IN transfected cells (p < 0.001)

Fig. 5

PPRV C protein inhibits the anti-viral effect via decreasing the production of exogenous IFN response. Monolayer HEK-293T cells in 6 well culture plates were transfected with pCMV-HA (2.5 µg) or pCMV-HA-C (2.5 µg). At 24 hpt, these cells were treated with IFN-β (1000 U/ml) for 24 h and then followed by infection with PPRV (1 MOI), VSV (0.1 MOI) and EMCV (0.1 MOI). At 24 hpi, all infected cells were harvested for RT-qPCR to measure the gene expression of PPRV H, EMCV VP1, and VSV L. a The expression of PPRV H in the transfected HA-C cells treated with IFN-β was significantly higher than that in transfected HA cells (p < 0.01); b The expression of EMCV VP1 in the transfected HA-C cells treated with IFN-β was significantly higher than that in transfected HA cells (p < 0.001); c The expression of VSV L in the transfected HA-C cells treated with IFN-β was significantly higher than that in transfected HA cells (p < 0.001)

Fig. 6

PPRV C protein inhibits the phosphorylation of STAT1. Monolayer HEK-293T cells in 6-well culture plates were transfected with pCMV-HA (2.5 µg) and pCMV-HA-C (2.5 µg), respectively. At 24 hpt, these cells were treated with IFN-β (1000 U/ml). At 24 h post treatment, cells were processed for the expression of total STAT1 and phosphorylated STAT1 with mouse anti-STAT1 mAb and rabbit anti-phospho-STAT1 (Tyr⁷⁰¹) mAb, respectively. Phosphorylated STAT1 expression was inhibited when C protein was expressed, with no effect on total STAT1. β-actin was used as a loading control. A representative image of the three independent experiments is shown here

Fig. 7

Mechanistic illustration of the interaction of PPRV C protein. General mechanism of PPRV entry, activation of various pathways, MAVS, JAK-STAT and IFN-β induction among others have been shown