Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4

Abstract

Viperin (virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible) is an interferon-inducible protein that mediates antiviral activity. Generally, rabies virus (RABV) multiplies extremely well in susceptible cells, leading to high virus titres. In this study, we found that viperin was significantly up-regulated in macrophage RAW264.7 cells but not in NA, BHK-21 or BSR cells. Transient viperin overexpression in BSR cells and stable expression in BHK-21 cells could inhibit RABV replication, including both attenuated and street RABV. Furthermore, the inhibitory function of viperin was related to reduce cholesterol/sphingomyelin on the membranes of RAW264.7 cells. We explored the up-stream regulation pathway of viperin in macrophage RAW264.7 cells in the context of RABV infection. An experiment confirmed that a specific Toll-like receptor 4 (TLR4) inhibitor, TAK-242, could inhibit viperin expression in RABV-infected RAW264.7 cells. These results support a regulatory role for TLR4. Geldanamycin, a specific inhibitor of interferon regulatory factor 3 (IRF3) (by inhibiting heat-shock protein 90 (Hsp90) of the IRF3 phosphorylation chaperone), significantly delayed and reduced viperin expression, indicating that IRF3 is involved in viperin induction in RAW264.7 cells. Taken together, our data support the therapeutic potential for viperin to inhibit RABV replication, which appears to involve upstream regulation by TLR4.

Figure 1

Viperin is induced in macrophage RAW264.7 cells during RABV infection. (A) Viperin levels as detected by Western blot in BHK-21, BSR, NA and RAW264.7 cell lines infected with rRC-HL at an MOI of 0.1 over time. RABV nucleoprotein (N) is defined as “N”. (B) Viperin/actin ratios over time in cell lines after rRC-HL infection.

Figure 2. Viperin expression inhibits RABV replication.

(A) Viperin inhibits RABV replication in viperin-eGFP-transfected BHK-21 cells. The viperin stably expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.1. Virus titres were determined at 24, 36, and 48 hpi. (B) RABV proteins in the infected viperin stably expressing BHK-21 cells were detected by Western blotting. (C) The N protein/actin, P protein/actin and M protein/actin ratios in Figure 2F were measured using Li-Cor Odyssey 3.0 analytical software version 29. (D) RNA expression levels of viperin. rRC-HL vRNA and N mRNA expression levels were detected by qRT-PCR at 24, 36, and 48 hpi. Viperin-expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.01. Data were normalized to β-actin expression and are presented as relative fold expression values to each control cell population infected with rRC-HL.

Figure 3. Viperin reduced cholesterol and sphingomyelin.

The BSR cells were transfected with pViperin or the pcDNA3.0 plasmid for 24 h, and cholesterol and sphingomyelin contents in BSR cells were measured using kits. Simultaneously, viperin was detected by Western blotting. The data represent averages of three independent experiments. The total amounts of cholesterol and sphingomyelin were determined using colorimetric assays.

Figure 4. Inhibition of cholesterol and sphingomyelin affected RABV budding and release.

(A) Effect of MβCD on BSR cell viability. BSR cells seeded in 96-well microplates were untreated or pre-treated with 0.5–8 mM MβCD for 1 h. The supernatants were removed and washed twice with PBS. An MTT assay was then performed. (B) Effect of MβCD on cholesterol and RABV replication. Cells seeded in six-well microplates were infected with rRC-HL at an MOI of 0.001 and then treated with 0.5–8 mM MβCD for 24 h at 37 °C. The supernatants were harvested for viral titration. The cells were assayed for cholesterol content using a cholesterol quantitation kit (40006; AAT Bioquest, Inc.) according to the manufacturer’s specfications. (C) Effect of MβCD on RABV replication. Based on A and B, a set of the cells was used to prepare cell lysates that were subjected to Western blot analysis for RABV N and β-actin protein expression. (D) The N protein/actin ratios in Fig. 3C were measured using Li-Cor Odyssey 3.0 analytical software version 29. The error bars were calculated from at least 3 independent inhibition tests. (E) Effect of myriocin on BSR cell viability. BSR cells were seeded in 96-well microplates and either untreated or pre-treated with 0.01–100 μm myriocin for 1 h. The supernatants were removed and washed twice with PBS. An MTT assay was then performed. (F) Effects of myriocin on sphingomyelin content and RABV replication. Cells seeded in six-well microplates were infected with rRC-HL at an MOI of 0.001 and then treated with 0.5–50 μm myriocin for 24 h at 37 °C. The supernatants were harvested for viral titration. The cells were assayed for sphingomyelin content using a sphingomyelin colorimetric assay kit (10009928). (G) Effect of myriocin on RABV replication. Based on D and E, another set of cells was used to prepare lysates that were then subjected to Western blotting analysis for RABV N and β-actin protein expression. (H) The N protein/actin ratios in Fig. 3G were measured using Li-Cor Odyssey 3.0 analytical software version 29.

Figure 5. TLR4 is involved in RABV-inducted viperin regulation.

(A) Altered TLR4 expression on membranes during RABV infection. RAW264.7 cells were mock-infected, UV-inactivated purified RABV or infected with rRC-HL at an MOI of 0.1 at 37 °C for 24 h, and cells were then analysed by flow cytometry. Data from triplicate experiments were then analysed using Guava soft 2.6 software. (B) RAW264.7 cells were infected with rRC-HL at an MOI of 0.1 and were then treated with 1.0 μM or 5.0 μM of TLR4-specific inhibitor TAK-242. The cell lysates were subjected to Western blotting analysis to detect viperin, RABV N protein, and β-actin protein expression. (C) The viperin protein/actin ratios in Figure 5D were measured using Li-Cor Odyssey 3.0 analytical software version 29. (D) The RAW264.7 cells were incubated with UV-inactivated purified RABV rRC-HL virion and were then treated with 5.0 μM of TLR4-specific inhibitor TAK-242. The cell lysates were subjected to Western blot analysis to detect viperin, RABV N protein, and β-actin protein expression. (E) The viperin/actin ratios in Fig. 5D were measured using Li-Cor Odyssey 3.0 analytical software version 29.

Figure 6. Identification of cellular factors in the TLR4 signal transduction pathway involved in RABV-inducted viperin regulation.

(A) Detection of cellular factors in the TLR4 signal transduction pathway involved in RABV-induced viperin regulation. RAW264.7 cells were infected with rRC-HL at an MOI of 0.1 and were then used to prepare lysates. These lysates were subjected to Western blotting analysis to detect MyD88, IRF3, IFN-β, viperin, NF-κB (p65), RABV N and β-actin protein expression. (B) Based on (A), the supernatants were used to assess IFN-α/β contents using ELISA kits. (C) The RAW264.7 cells were treated with 2,000 pg/mL IFNβ for 24 h, and the cell lysates were then subjected to Western blotting analysis to detect viperin protein expression.

Figure 7. IRF3/HSP90 rather than NF-κB participates in viperin regulation.

(A) In vitro infection with RABV affecting IRF3 expression. RAW264.7 cells were infected with rRC-HL at an MOI of 0.1, and DMSO (for dissolving geldanamycin [GA], an inhibitor of IRF3) was added as a mock control. The cell lysates were prepared and subjected to Western blotting analysis to detect IRF3, Hsp90, viperin, RABV N, and β-actin protein expression. (B) GA inhibits IRF3 and viperin expression. RAW264.7 cells were infected with rRC-HL at an MOI of 0.1 and were treated with 1.0 μM GA. The cell lysates were prepared and subjected to Western blotting analysis to detect IRF3, Hsp90, viperin, RABV N, and β-actin protein expression. (C) The IRF3 and viperin protein/actin ratios in Fig. 3A,B were measured using Li-Cor Odyssey 3.0 analytical software version 29. (D) Mock-treated RAW264.7 cells were infected with rRC-HL at an MOI of 0.1. The cell cultures were used to prepare lysates and were subjected to Western blotting analysis to detect NF-κB (p65), viperin, RABV N, and β-actin protein expression. (E) RAW264.7 cells were infected with rRC-HL at an MOI of 0.1 and then treated with the NF-κB (p65)-specific inhibitor BAY11-7082 to a final concentration of 2.0 μM. The cell cultures were used to prepare lysates and were subjected to Western blotting analysis to detect NF-κB (p65), viperin, RABV N, and β-actin protein expression. (F) The NF-κB and viperin protein/actin ratios in Fig. 3D, E were measured using Li-Cor Odyssey 3.0 analytical software version 29.

Figure 8. Proposed model of viperin-mediated inhibition of RABV replication and its up-stream signal regulation.

The novel data from this study are hypothesized in this figure.