The liver X receptor agonist LXR 623 restricts flavivirus replication

Abstract

The vector-borne flaviviruses (VBFVs) are well known for causing great misery and death in humans worldwide. The VBFVs include those transmitted by mosquitos, such as Zika virus (ZIKV), dengue virus; and those transmitted by ticks including the tick-borne flavivirus serocomplex and Powassan virus (POWV). Two of our recent reports showed that intracranial POWV infection in the reservoir host, Peromyscus leucopus, was restricted and caused no overt clinical disease. Several modes of analyses suggested activation of the LXR pathway. Activation of the LXR pathway leads to increased efflux of cholesterol from cells and consequent disturbances in membrane biogenesis. Because VBFV replication is dependent on membrane biogenesis, we evaluated the effect of an LXR agonist (LXR623) on POWV and ZIKV infection and observed that the compound impaired permissive replication of both viruses in a human neuroblastoma SK-N-SH cell line. The LXR agonist resulted in failure of the viruses to induce ER expansion and elaborate vesicle formation, suggesting that the efflux of cholesterol was part of the antiviral mechanism. We also observed that the LXR agonist contributed to the mechanism of virus suppression by increased expression of mRNAs encoding for the antiviral cytokines CXCL10, RANTES and IFN1β. In sharp contrast, a LXR antagonist (GSK2033) had no significant effect on VBFV replication. We conclude that LXR623 impairs flavivirus replication by stimulating cellular antiviral factors.

Keywords: Flavivirus; LXR 623; Powassan virus; Zika virus; liver X receptor; virus restriction.

Figure 1.

Analysis of the effect of LXR 623 on POWV replication. (A) Results from RNASeq suggesting that the LXR pathway is upregulated in Peromyscus leucopus mouse brains infected with POWV. The graph was drawn from the original dataset described in Mlera et al. [18] and the mRNA expression levels were in comparison to mock-infected brains. (B) Evaluation of cholesterol efflux from SK-N-SH cells treated with LXR 623. (C) SK-N-SH cells were infected with POWV at a MOI of 0.01 in the presence of the indicated compounds and supernatants collected at different time points for titration by immunofocus assay. The values represent means from 3 biological replicates and the error bars represent SD. ns, not significant; **, p < 0.01 (multiple t-tests).

Figure 2.

ZIKV infection upregulates LXR-α late in infection and LXR 623 negatively impacts ZIKV replication. (A) SK-N_SH cells were mock-infected or infected with ZIKV at a MOI of 1 and cell lysates collected over a 4-day time course. Western blot analysis was performed to detect changes in LXR-α expression. (B). Cells were seeded at 2 × 10⁶ cells/T75 flask, incubated overnight and infected at a MOI of 0.01 the next day. Infected cells were incubated with 50 µM LXR 623 for 48 h at 37°C and 5% CO₂. Uninfected controls were incubated in EMEM. Replication kinetics of ZIKV MR766 in cells incubated in EMEM, DMSO, 50 or 100 µM LXR 623, or 50 or 100 µM GSK 2033 are shown. (C) Replication kinetics of ZIKV Paraiba in cells incubated in EME, DMSO, 50 or 100 µM LXR 623, or 50 or 100 µM GSK 2033. (D) Analysis of the effect of LXR 623 treatment at 10, 20 or 30 µM LXR 623. *p < 0.05. Evaluation of the impact of LXR 623 added to infected cell culture post infection. Infections were done at a MOI of 0.01 (E) or a high MOI of 10 (F). The inoculum was incubated with cells for 1 h followed by washing twice with PBS and replacement with drug-containing media. Values represent means from 3 biological replicates and error bars indicate SD from the mean. ****p < 0.0001; ns, not significant (multiple t-tests).

Figure 3.

TEM of SK-N-SH cells infected with ZIKV in the presence or absence of LXR 623. SK-N-SH cells were seeded at 1 × 10⁵ cells/well on Thermanox coverslips in EMEM, DMSO, 50 uM LXR 623, or 50 uM GSK 2033. After 24 h treatment, cells were mock or virus infected (POWV LB or ZIKV MR766) at a MOI of 10, fixed, resin embedded, 70 nm section cut, and processed for TEM. The arrows point to ER expansion caused by flavivirus infection and the DMSO control represent the “classical” expansion. LXR 623 treatment in POWV infection led to failed ER expansion, but ZIKV induced some distorted expansion. Typical ER expansion was observed in POWV infection exposed to GSK 2033 and distorted membranes were also observed in ZIKV infection. Scale bar = 800 nm.

Figure 4.

The LXR agonist GW3965 does not affect ZIKV replication. SK-N-SH cells were treated with 2 μM GW3965 then infected as described in Figure 2.

Figure 5.

LXR 623 downregulates IFN-β transcripts. Uninfected SK-N-SH cells were treated with DMSO or LXR 623 at 50 μM. Cells were harvested at 24 h post treatment and total RNA was extracted. cDNA was synthesized and subjected to qPCR. IFN-β transcript expression levels were normalized to HPRT transcripts. Values were from 3 biological replicates and error bars represent SD. ***, p < 0.001; ****, p < 0.0001; ns, not significant (two tailed t-test).

Figure 6.

Effect of LXR 623 on the expression levels of cytokine-encoding mRNAs in ZIKV-infected SK-N-SH cells. Cells were infected at a MOI of 10, and transcript expression levels were normalized to GAPDH. Values represent the mean from of 3 biological replicates and error bars indicate SD from the mean. ***p < 0.001; ****p < 0.0001; ns, not significant.

Figure 7.

Protective effect of LXR 623 on ZIKV-infected SK-N-SH cells from apoptosis. Cells were pre-treated with DMSO, GSK2033 or LXR 623 followed by infection at a MOI of 0.01 and allowed to grow for 4 dpi. At 4 dpi, culture media was aspirated, and cells washed in PBS followed by fixing with 4% paraformaldehyde for 10 min. The fixative was removed, and cells were rinsed in PBS 3 times and then stained with Coomassie Brilliant Blue for 2 min. The stain was removed followed by 3 washes in PBS. Stained cells were imaged using a 40× objective on an AxioVert.A1 microscope (Zeiss) equipped with an Axiocam 503 mono camera.

Figure 8.

Proliferation of SK-N-SH cells in LXR 623. (A) SK-N-SH cells were seeded at 10⁴ cells/well. Proliferation was evaluated over the course of 4 days. (B) Cells were seeded at 5 × 10⁴ cells/well and the assay was done at 72 h post treatment. Values represent the mean from of 3 biological replicates and error bars indicate SD from the mean. ***p < 0.001; ****p < 0.0001 (multiple t-test).