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. 2016 Sep;161(9):2491-501.
doi: 10.1007/s00705-016-2950-4. Epub 2016 Jun 29.

The LXR ligand GW3965 inhibits Newcastle disease virus infection by affecting cholesterol homeostasis

Xiang-Xiang Sheng  1 , Ying-Jie Sun  2 , Yuan Zhan  2 , Yu-Rong Qu  2 , Hua-Xia Wang  1 , Miao Luo  1 , Ying Liao  2 , Xu-Sheng Qiu  2 , Chan Ding  3 , Hong-Jie Fan  1 , Xiang Mao  4   5
Affiliations

The LXR ligand GW3965 inhibits Newcastle disease virus infection by affecting cholesterol homeostasis

Xiang-Xiang Sheng et al. Arch Virol. 2016 Sep.

Abstract

Newcastle disease (ND) is a contagious disease that affects most species of birds. Its causative pathogen, Newcastle disease virus (NDV), also exhibits considerable oncolytic activity against mammalian cancers. A better understanding of the pathogenesis of NDV will help us design efficient vaccines and novel anticancer strategies. GW3965, a widely used synthetic ligand of liver X receptor (LXR), induces the expression of LXRs and its downstream genes, including ATP-binding cassette transporter A1 (ABCA1). ABCA1 regulates cellular cholesterol homeostasis. Here, we found that GW3965 inhibited NDV infection in DF-1 cells. It also inhibited NF-κB activation and reduced the upregulation of proinflammatory cytokines induced by the infection. Further studies showed that GW3965 exerted its inhibitory effects on virus entry and replication. NDV infection increased the mRNA levels of several lipogenic genes but decreased the ABCA1 mRNA level. Overexpression of ABCA1 inhibited NDV infection and reduced the cholesterol content in DF-1 cells, but when the cholesterol was replenished, NDV infection was restored. GW3965 treatment prevented cholesterol accumulation in the perinuclear area of the infected cells. In summary, our studies suggest that GW3965 inhibits NDV infection, probably by affecting cholesterol homeostasis.

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Figures

Fig. 1
Fig. 1
The effects of GW3965 on NDV infection. (A) The DF-1 cells were cultured with different concentrations of GW3965. Cell viability was determined by MTT assay at 24 h. (B) Synthetic LXR agonist GW3965 inhibited NDV infection in DF-1 cells. Cells were pretreated with GW3965 (1 μM) for 2 h, then infected with NDV (MOI = 1). GW3965 (1 μM) was continuously present during the infection. The expression level of the viral NP protein was determined by western blot. (C) The titers of the supernatant were determined by TCID50. (D) The viral gRNA in cells was detected by qRT-PCR assay
Fig. 2
Fig. 2
GW3965 affects NDV entry and replication. (A) GW3965 did not affect NDV attachment. DF-1 cells were incubated with NDV (MOI = 1) at 4 °C for 1 h in the presence of GW3965 (1 μM). The viral gRNA copy number was determined by qRT-PCR assay. (B) GW3965 inhibited NDV entry. DF-1 cells were first incubated with NDV (MOI = 1) at 4 °C for 1 h and were then cultured at 37 °C in the presence of GW3965 (1 μM) for 1 h. The cells were washed three times. The viral gRNA copy number was determined by qRT-PCR. (C) GW3965 inhibited NDV replication. After infected with NDV (MOI = 1), DF-1 cells were incubated with GW3965 (1 μM). Viral gRNA copy numbers were determined by qRT-PCR at 8 hpi and 10 hpi
Fig. 3
Fig. 3
GW3965 inhibits NDV-induced NF-κB activation. (A) Cells were infected with NDV (MOI = 1) in the absence or presence of GW3965 (0.1, 0.5 and 1 μM) for 12 h. The protein levels of the NF-κB pathway were analyzed by Western blot with specific antibodies. β-actin was used as the loading control. (B and C) Quantifications of Western blots were performed with ImageJ software
Fig. 4
Fig. 4
GW3965 reduces mRNA and protein expression levels of the proinflammatory cytokines in infected DF-1 cells. DF-1 cells were infected by NDV (MOI = 1) and incubated with GW3965 (1 μM). The cells were collected at 12 h post-treatment. The relative expression level of (A) IL-1β, (B) IL-6, (C) IL-8, (D) TNF-α and (E) IFN-β was determined by qRT-PCR. Values were normalized to internal control β-actin. The protein concentrations of (F) IL-1β, (G) IL-6, (H) IL-8, (I) IFN-β and (J) TNF-α in the supernatant were determined by ELISA assay
Fig. 5
Fig. 5
NDV infection disturbs LXR and its downstream lipogenic gene expression. (A-B) The effect of the NDV infection on the expression of lipogenic genes. DF-1 cells were infected with NDV (MOI = 1). Total RNA was extracted at 6, 12, and 24 hpi. The mRNA levels of LXR-α, SREBP-1, PPAR-γ1, FASN, and ABCA1 were determined. (C-G) The effect of GW3965 on the expression of lipogenic genes in infected DF-1 cells. DF-1 cells were infected with NDV (MOI = 1) in the presence or absence of GW3695 (1 μM) for 12 h. The relative mRNA levels of LXR-α, SREBP-1, PPAR-γ1, FASN, and ABCA1 were determined by qRT-PCR. The RNA copy number in NDV-infected cells was considered to be 1
Fig. 6
Fig. 6
ABCA1 is involved in the inhibition of NDV infection. (A) Overexpression of ABCA1 inhibited NDV infection, whereas cholesterol replenishment restored NDV infectivity. DF-1 cells were transfected with EGFP (lane 1) or EGFP-ABCA1 (lane 2) for 24 h before the infection. The EGFP-ABCA1 over-expressing cells were incubated with 400 μg/ml cholesterol during the infection (lane 3). NDV-NP protein expression levels were determined at 12 hpi by Western blot. (B) Cholesterol contents were determined in mock-infected and NDV-infected cells that were previously transfected with EGFP or EGFP-ABCA1 at 12 hpi. (C) Cholesterol replenishment restored NDV infectivity in GW3965 (1 μM)-treated cells at 12 hpi. (D) Cholesterol contents were determined in mock-infected and NDV-infected cells (treated and non-treated by 1 μM GW3965) at 12 hpi. GW3965 treatment reduced the cholesterol content in DF-1 cells. (E) The cellular HMGCR mRNA level was determined by qRT-PCR in infected cells at different time points. (F) DF-1 cells were either mock-infected or infected with NDV (MOI = 1) in the presence or absence of GW3965. The cells were then fixed and stained with filipin III (0.05 mg/mL) at 12 hpi
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