The Oxysterol 25-Hydroxycholesterol Inhibits Replication of Murine Norovirus

Abstract

Cholesterol, an essential component of mammalian cells, is also an important factor in the replicative-cycles of several human and animal viruses. The oxysterol, 25-hydroxycholesterol, is produced from cholesterol by the enzyme, cholesterol 25-hydroxylase. 25-hydroxycholesterol (25-HC) has been shown to have anti-viral activities against a wide range of viruses, including a range of positive-sense RNA viruses. In this study, we have investigated the role of 25-HC in norovirus replication using murine norovirus (MNV) as a model system. As a control, we employed herpes simplex virus-1 (HSV-1), a pathogen previously shown to be inhibited by 25-HC. Consistent with previous studies, 25-HC inhibited HSV-1 replication in the MNV-susceptible cell line, RAW264.7. Treating RAW264.7 cells with sub-cytotoxic concentrations of 25-HC reduced the MNV titers. However, other sterols such as cholesterol or the oxysterol, 22-S-hydroxycholesterol (22-S-HC), did not inhibit MNV replication. Moreover, treating MNV-infected RAW264.7 cells with 25-HC-stimulated caspase 3/7 activity, which leads to enhanced apoptosis and increased cell death. Our study adds noroviruses to the list of viruses inhibited by 25-HC and begins to offer insights into the mechanism behind this inhibition.

Keywords: 25-HC; Apoptosis; MNV; Murine norovirus; Nystatin; Replication.

Figure 1

In vitro anti-viral activity of 25-HC on MNV replication. (A) RAW264.7 cells were infected with MNV at a multiplicity of infection (MOI) of approximately 0.05 TCID₅₀/cell and were immediately treated with a range of concentrations of 25-HC (up to 135 µM) or nystatin (up to 500 µM) or the ethanol-only control. At regular intervals, replicate wells were harvested, the supernatant clarified by centrifugation and the virus titer determined by TCID₅₀ assay. Data show mean virus titer (n = 2 ± SEM). (B) RAW264.7 cells were incubated with the indicated compounds for 72 h before the cell viability was measured by MTS assay. The ethanol (EtOH) or dimethyl sulphoxide (DMSO) solvents were used as controls. Data are expressed as mean percentage cell viability and normalized to untreated cells (n = 3 ± SEM).

Figure 2

The oxysterol, 25-HC, reduced the titer of MNV. RAW264.7 were infected with MNV at a MOI of approximately (A) 0.05 TCID₅₀/cell or (B) 5 TCID₅₀/cell and immediately treated with 25-HC (6.6 µM), nystatin (50 µM) or ethanol-only control. At regular intervals, replicate wells were harvested, supernatant clarified by centrifugation and virus titer determined by TCID₅₀ assay. Data show mean virus titer (n = 3 ± SEM, * = p < 0.05, ** = p < 0.01, *** = p < 0.001). (C) RAW264.7 cells were infected with MNV and immediately treated with 25-HC (6.6 µM), nystatin (50 µM) or ethanol-only control. At 12 h post-infection, the expression of MNV NS1/2 protein was detected by indirect immunofluorescence and the number of virus positive cells was quantified using an IncuCyte ZOOM FLR. Data show the mean number of virus-infected cells per well and statistical analysis compared to ethanol-only control (n = 3 ± SEM, *** = p < 0.001).

Figure 3

Pre-treating RAW264.7 cells with 25-HC inhibits MNV replication. RAW264.7 cells were pre-treated with 25-HC (6.6 µM), nystatin (50 µM) or the ethanol-only control at 24, 16 or 4 h before infection (−24 h, −16 h or −4 h, respectively), and the compounds were removed prior to the addition of MNV at (A) 0.05 TCID₅₀/cell or (B) 5 TCID₅₀/cell. Subsequently, the cells were infected with MNV and immediately treated with the compounds (0 h) or treated at 4 h post-infection (+4 h) after removal of the virus. At 48 h post-infection, the supernatants were removed, clarified by centrifugation and production of infectious virus determined by TCID₅₀ assay on RAW264.7 cells. Data show mean virus titer and statistical analysis of 25-HC by comparison to the ethanol-only control (n = 3 ± SEM, * = p < 0.05, ** = p < 0.01).

Figure 4

MNV-induced apoptosis in the presence of 25-HC. Replicate wells of RAW264.7 cells were infected with MNV at an MOI of (A) 0.05 or (B) 5 TCID₅₀/cell and immediately treated with 25-HC (6.6 µM). As controls, the replicate wells were infected with MNV without the addition of 25-HC or treated with 25-HC alone. To quantify apoptosis, the caspase 3/7 assay reagent was added directly to the wells immediately after infection and the caspase activity was measured by fluorescence at regular intervals using IncuCyte ZOOM FLR. Data are shown as mean caspase-3/7 positive cells per well and statistical analysis of MNV with 25-HC by comparison to the MNV-only sample (n = 4 ± SEM, * = p < 0.05, ** = p < 0.01).

Figure 5

The effect of 25-HC is oxysterol-specific. (A) RAW264.7 cells were infected with MNV at 0.05 TCID50/cell in the presence of 25-HC, 22-S-HC, cholesterol (all 6.6 µM), nystatin (50 µM) or ethanol-only control. Cell viability was measured by MTS assay 72 h post-infection. Data are expressed as mean percentage cell viability normalized to untreated and uninfected cells (n = 3 ± SEM, *** = p < 0.001). (B) Replicate wells of RAW264.7 cells were infected with HSV-1–GFP at an MOI of 0.03 PFU/cell and treated with 25-HC, 22-S-HC, cholesterol (all 6.6 µM), nystatin (50 µM) or ethanol-only control. Cells were incubated for 48 h before GFP expression was quantified using an Incucyte ZOOM FLR. Data show the mean number of HSV-1-positive cells per well normalized to ethanol solvent control and statistical analysis compared to ethanol-only control (n = 3 ± SEM, * = p < 0.05, ** = p < 0.01).