Melatonin inhibits bovine viral diarrhea virus replication by ER stress-mediated NF-κB signal pathway and autophagy in MDBK cells

Abstract

Bovine viral diarrhea-mucosal disease (BVD-MD) is a contagious disease in cattle, caused by the bovine viral diarrhea virus (BVDV). This virus continues to spread globally, exerting pressure on both public health and the economy. Despite its impact, there are currently no effective drugs for treating BVDV. This study utilized Madin-Darby bovine kidney (MDBK) cells as a model to investigate the antiviral effects of melatonin against Bovine Viral Diarrhea Virus (BVDV) and its connection with endoplasmic reticulum (ER) stress. Our results show that melatonin can suppress BVDV proliferation in MDBK cells by modulating the endoplasmic reticulum (ER) stress-mediated NF-κB pathway and autophagy. Specifically, melatonin alleviated ER stress, inhibited the activation of IκBα and p65, regulated autophagy, and reduced the expression levels of pro-inflammatory cytokines. Further, when we treated BVDV-infected cells with the ER stress inducer thapsigargin, it led to significant activation of the NF-κB pathway and autophagy. Conversely, treating the cells with the ER stress inhibitor 4-phenylbutyric acid reversed these effects. These findings suggest that melatonin exerts its antiviral effects primarily through the PERK-eIF2α-ATF4 of ER stress-mediated NF-κB pathway and autophagy. Overall, our study underscores the potential of melatonin as an effective protective and therapeutic option against BVDV, offering insights into its anti-infective mechanisms.

Keywords: BVDV; NF-κB pathway; PERK-eIF2α-ATF4; autophagy; melatonin.

Figure 1

MT downregulated the replication level of BVDV. (A) Cytotoxicity of 0.25-5 mM MT in MDBK cells at 24 and 36 (h) (B) In BVDV-infected MDBK cells treated with 100, 250, or 500 μM MT, RT-qPCR was performed to measure the levels of BVDV mRNA expression at 24 and 36 (h) (C) The BVDV E2 protein expression levels at 24 and 36 h post-infection after treatment with 100, 250, or 500 μM MT. (D) The IFA image of BVDV E2 protein (green), DAPI (blue). The scale bar represents 100 μm. Data are presented as means ± SEM of 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2

MT inhibited BVDV attachment and replication. (A) The schematic of DMBK cells treatment with 500 µM MT and BVDV infection in the inactivation, attachment, attachment, replication, and release processes. (B) RT-qPCR analyzed the viricidal effect of 500 μM MT on BVDV virions. The BVDV mRNA expression levels in MDBK cells with 500 μM MT treatment of BVDV replication cycle including attachment (C), internalization (D), replication (E), and release (F). Data are presented as means ± SEM of 3 independent experiments. *, P < 0.05; ***, P < 0.001; ns, not significant.

Figure 3

MT suppressed the BVDV−induced PERK-eIF2α-ATF4 pathway of ER stress in MDBK cells. (A) The expression levels of GRP78, PERK, p-elF2α, elF2α, ATF4, and CHOP protein were detected by western blot at 24 (h) (B) The IFA image of ER-tracker (red), and DAPI (blue). Scale bars represent 25 μm. Data are presented as means ± SEM of 3 independent experiments. **, P < 0.01; ***, P < 0.001; ns, not significant.

Figure 4

MT regulated autophagy in BVDV infection of MDBK cells. (A) p-p65, p-IκB, p65, IκB expression were analyzed by western blot. (B) p65 (green) immunofluorescence staining was used to observe p65 nuclear translocation. DAPI: blue, nucleus. Scale bar, 10 μm. (C) LC3 II, p62 expression were analyzed by western blot. (D) LC3 (green) immunofluorescence staining was used to observe autophagy. DAPI: blue, nucleus. Scale bar, 10 μm. Data are presented as means ± SEM of 3 independent experiments. *, P < 0.05,**, P < 0.01, ***, P < 0.001.

Figure 5

The PERK-eIF2α-ATF4 pathway of ER stress participated in the mechanisms of MT against BVDV replication in MDBK cells. Western blot detected the expressions of GRP78, PERK, p-elF2α, elF2α, ATF4, and CHOP protein in BVDV-infected MDBK cells with MT, TG (A), or 4-PBA (B) treatment for 24 (h) (C) BVDV mRNA expression levels in MDBK cells with BVDV infection and treated with MT, TG, or 4-PBA for 24 (h) (D, E) The IFA image of BVDV E2 protein (green), DAPI (blue). The scale bar represents 50 μm. Data are presented as means ± SEM of 3 independent experiments. *, P < 0.05,**, P < 0.01, ***, P < 0.001; ns, not significant.

Figure 6

MT ameliorated PERK-eIF2α-ATF4 pathway of ERS mediated NF-κB signal pathway and autophagy in BVDV-infected MDBK cells. The expression levels of p-IκB, IκB, p-p65, and p65 protein detected by western blot in BVDV-infected MDBK cells with MT, TG (A), or 4-PBA (B) treatment for 24 (h) The LC3 II and p62 protein expression levels in MDBK cells with BVDV infection and MT, TG (C), or 4-PBA (D) treatment for 24 (h) Data are presented as means ± SEM of 3 independent experiments. *, P < 0.05,**, P < 0.01, ***, P < 0.001.

Figure 7

MT alleviated BVDV-induced inflammation in MDBK cells. The relative mRNA expressions of IL-1β (A), IL-6 (B), IL-8 (C), IL-18 (D), and TNF-α (E) in the BVDV-infected group, BVDV-infected group treated with MT (250, and 500 μM), or DMSO, and untreated group were detected by RT-qPCR at 24 h GAPDH gene mRNA level set as a standardized internal control. Data are presented as means ± SEM of 3 independent experiments. **, P < 0.01, ***, P < 0.001.

Figure 8

Schematic diagram of MT inhibiting BVDV replication via ER stress-mediated NF-κB pathway and autophagy. BVDV infection activates the PERK-eIF2α-ATF4 branch of the ER stress, promotes p65 phosphorylation and nuclear translocation, and regulates autophagy. MT exerts its anti-BVDV effect via ameliorating the BVDV-induced NF-κB signaling pathway and autophagy through inhibiting the PERK- eIF2α-ATF4 pathway.