Proteasome Inhibitor PS-341 Effectively Blocks Infection by the Severe Fever with Thrombocytopenia Syndrome Virus

Abstract

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever disease caused by SFTSV, a newly discovered phlebovirus that is named after the disease. Currently, no effective vaccines or drugs are available for use against SFTSV infection, as our understanding of the viral pathogenesis is limited. Bortezomib (PS-341), a dipeptide-boronic acid analog, is the first clinically approved proteasome inhibitor for use in humans. In this study, the antiviral efficacy of PS-341 against SFTSV infection was tested in human embryonic kidney HEK293T (293T) cells. We employed four different assays to analyze the antiviral ability of PS-341 and determined that PS-341 inhibited the proliferation of SFTSV in 293T cells under various treatment conditions. Although PS-341 did not affect the virus absorption, PS-341 treatment within a non-toxic concentration range resulted in a significant reduction of progeny viral titers in infected cells. Dual-luciferase reporter assays and Western blot analysis revealed that PS-341 could reverse the SFTSV-encoded non-structural protein (NS) mediated degradation of retinoic acid-inducible gene-1 (RIG-I), thereby antagonizing the inhibitory effect of NSs on interferons and blocking virus replication. In addition, we observed that inhibition of apoptosis promotes virus replication. These results indicate that targeting of cellular interferon pathways and apoptosis during acute infection might serve as the bases of future therapeutics for the treatment of SFTSV infections.

Keywords: Apoptosis; Bortezomib; IFN-β; PS-341; Severe fever with thrombocytopenia syndrome virus (SFTSV).

Fig. 1

Toxicity and efficacy of PS-341 in 293T cells. A 293T cells were each treated with 0, 6.25, 12.5, 25, 50, 100, 200, 400, 600 or 800 nmol/L PS-341, and analyzed for survival using the MTT assay at 24 h. B 293T cells were treated with PS-341 as described in the schematic diagram. C With SFTSV (MOI=0.1) infection, 293T cells were each treated with 0, 25, 50, 100, or 200 nmol/L PS-341 for 24 h under four treatment conditions. The infected cells were collected, and measured by qRT-PCR. Asterisk indicates significant difference in viral RNA level between PS-341 untreated group and PS-341 treated group, **P < 0.01, ***P < 0.001. D 293T cells were incubated with SFTSV (MOI 0.01, 0.1, 1, 2 or 4) for 1 h then and cultured in medium containing DMSO (Mock) or PS-341 (50 nmol/L) for 24 h. The viral RNA in the whole cell lysate was extracted for qRT-PCR analysis. Asterisk indicates significant difference in viral RNA level between the mock group and PS-341 treated group at the same virus infected titer, **P < 0.01, ***P < 0.001. E 293T cells were infected with SFTSV (MOI = 1) and treated with PS-341 (50 nmol/L) under four treatment conditions for 24 h and detected by immunofluorescence with rabbit anti-NP primary antibody and followed by secondary antibody labeled with TRITC. DAPI was used to counterstain the nuclei. The histogram shows the statistical analysis of 293T cell fluorescence image (Bar = 30 μm). Asterisk indicates significant difference in viral RNA level between Mock and PS-341 -treated group, **P < 0.01. F The total protein of cells was extracted and detected by Western blotting. Ctrl: uninfected cells; Mock: SFTSV-infected cells. All samples were run in triplicate, and the experiment was repeated three times.

Fig. 2

The effect of PS-341 on SFTSV infectivity and absorption. SFTSV (MOI = 0.1) was pre-treated with 50 nmol/L PS-341 or DMSO (Mock) for 0, 2, 4, or 8 h p.i. at 37 °C, and 293T cells were infected with the mixture for 1 h., After washed three times with DMEM, fresh medium were added. The cells (A) and supernatant (B) were collected at 24 h for qRT-PCR analysis. Asterisk indicates significant difference in viral RNA level between mock and PS-341 treated group at the same incubation time point, *P < 0.05, **P < 0.01, ***P < 0.001. C 293T cells were co-cultured with 50 nmol/L PS-341 and SFTSV (MOI = 0.1) or with only SFTSV for 30 min at 4 °C. The cells and supernatants were collected for qRT-PCR detection. n.s., P > 0.05. All samples were run in triplicate, and the experiment was repeated three times.

Fig. 3

The inhibitory effect of PS-341 on SFTSV replication and release. 293T cells were PS-341 (50 nmol/L)-treated under four conditions, the infected cells (A) and culture supernatants (B) were collected at particular points of time, and analyzed by qRT-PCR. The relative viral RNA level was expressed as fold change relative to the value of viral RNA at 0 h (set as 1). Asterisk indicates significant difference in viral RNA level between Mock and PS-341-treated group, **P < 0.01, ***P < 0.001. C, D After SFTSV absorption for 1 h, PS-341 (50 nmol/L) was added to 293T cells at 4, 8, 12, and 24 h p.i. The infected cells (C) and supernatants (D) were collected at 48 h p.i. for qRT-PCR detection. Asterisk indicates significant difference in viral RNA level between Mock and PS-341-treated group. *P < 0.05, **P < 0.01, ***P < 0.001. All samples were run in triplicate, and the experiment was repeated three times.

Fig. 4

PS-341 suppresses the NSs-mediated degradation of RIG-I. A After infected with SFTSV (MOI = 1), 293T cells were infected with SeV (20 HA/mL) and treated with DMSO or PS-341 (50 nmol/L). Then the cells were collected at 24 h, the mRNA levels of IFN-β were analyzed by qRT-PCR. The relative IFN-β RNA level was expressed as fold change relative to the value of IFN-β RNA in mock group (set as 1). B 293T cells (5 × 10⁵) were co-transfected with the IFN-β luciferase reporter plasmid (200 ng), pRL-SV40 plasmid (1 ng), and NSs-HA plasmid (800 ng) or VR1012 plasmid (800 ng). After 24 h, cells were infected and incubated with SeV (20 HA/mL) for another 16 h, and then luciferase activities were detected. C 293T cells (5 × 10⁵) were transfected with the same IFN-β luciferase reporter plasmid (200 ng), pRL-SV40 plasmid (1 ng), RIG-I-Flag plasmid (400 ng), NSs-HA plasmid (400 ng) or VR1012 plasmid. After a 48 h culture period, luciferase activities were detected. D 293T cells (5 × 10⁵) were transfected with NSs-HA plasmid (500 ng) and then treated with DMSO or PS-341 for 24 or 48 h and detected the expression of NSs-HA by Western blotting. E, F 293T cells were also transfected with the same plasmids as (C), and after 24 h, PS-341 was added to cells before qRT-PCR test (E) or detected the luciferase activities (F). The relative IFN-β RNA level was expressed as fold change relative to the value of IFN-β RNA in Mock group (set as 1). The expression level of proteins was determined by immunoblotting analysis. All samples were run in triplicate, and the experiment was repeated three times. *P < 0.05, **P < 0.01.

Fig. 5

PS-341 inhibits the replication of SFTSV by inducing apoptosis. A, B 293T and Vero cells were each treated with 50 nmol/L PS-341 under four conditions, the infected cells (A) and supernatants (B) were collected at 24 h, and analyzed by qRT-PCR. The virus RNA level in PS-341 untreated group was set as 100%, the inhibition rate of PS-341 to SFTSV was calculated and plotted under four conditions. C 293T cells and D Vero cells were infected with SFTSV (MOI = 0.1) and treated with PS-341 (50 nmol/L) and STS (200 nmol/L) or PS-341 (50 nmol/L) and Ac-YVAD-CHO (40 µmol/L) for 24 h, and then collected for qRT-PCR analysis. All samples were run in triplicate, and the experiment was repeated three times. *P < 0.05.