Research ArticleVIROLOGY

Zika virus degrades the ω-3 fatty acid transporter Mfsd2a in brain microvascular endothelial cells and impairs lipid homeostasis

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Science Advances  23 Oct 2019:
Vol. 5, no. 10, eaax7142
DOI: 10.1126/sciadv.aax7142
  • Fig. 1 ZIKV infection decreases Mfsd2a at the protein level both in vitro and in vivo.

    (A and B) hBMECs were challenged with ZIKV SZ01 or PRVABC59 at the indicated MOI for 48 hours (A) or were infected with ZIKV SZ01 at an MOI of 1 for various time courses (B). A representative Western blot analysis (of n = 3 independent experiments) is demonstrated. The expression of Mfsd2a, ZIKV E, and β-actin was assessed. (C) hBMECs were challenged with SZ01 or PRVABC59, and viral RNA (vRNA) and Mfsd2a mRNA were determined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Data are presented as means ± SD of n = 3 experiments run with duplicate samples. n.s., not significant. (D) A549 stable cells expressing Mfsd2a-GFP were infected with ZIKV strains. Immunofluorescent (IF) staining with ZIKV E antibody was performed using the indicated antibodies. Scale bar, 20 μm. (E to G) The neonatal BALB/c mice were infected with ZIKV via intracerebral injection with 10 or 100 plaque-forming units (PFU). The mice were euthanized at 11 days post-infection (dpi) to isolate the brain tissues. The brain morphology (E) (scale bar, 1 cm), body weights and brain weights (F), protein levels of Mfsd2a and ZIKV E in the brain (G), and Mfsd2a mRNA level (H) were measured by weighing, qRT-PCR, or Western blotting. PBS, phosphate-buffered saline. (I and J) Representative IF images (of n = 4 mice per treatment) of mouse brain hippocampus dentate gyrus serial pathological section by staining for ZIKV E, Mfsd2a, the endothelial cell marker CD31, and the nuclei with DAPI (4′,6-diamidino-2-phenylindole) in the infected or control brains. Scale bars, 200 μm. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by one-way analysis of variance (ANOVA). Photo credit: Jia Zhou, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College.

  • Fig. 2 ZIKV E protein disrupts Mfsd2a expression.

    (A) HEK293T cells were cotransfected with plasmids encoding Mfsd2a-Myc and either ZIKV structural C/E or nonstructural NS1/2A/3/4A/4B/5 FLAG fusion proteins. The expression of Mfsd2a-Myc and individual ZIKV proteins was analyzed by Western blotting at 48 hours after transfection. (B) A549 stable cells expressing Mfsd2a-GFP were transfected with a ZIKV E–FLAG plasmid or an empty vector control for 48 hours. Representative GFP and ZIKV E IF confocal images (n = 3 per treatment) were captured. DAPI staining indicates the nuclei. Scale bar, 40 μm. (C) hBMECs or JEG-3 cells were transfected with various amounts of the ZIKV E–Flag plasmid or empty vector control for 48 hours, and the expression of endogenous Mfsd2a was measured by Western blot. (D) An Mfsd2a-Myc plasmid was cotransfected with one of the plasmids encoding ZIKV E–Flag, WNV E–Flag, or HCV E1E2 into HEK293T cells. The expression of Mfsd2a-Myc and individual viral envelope proteins was determined by Western blotting at 48 hours after transfection.

  • Fig. 3 ZIKV E protein targets Mfsd2a for degradation.

    (A) Pull-down assay using recombinant His-tagged ZIKV E with the cell lysates containing transiently expressed Mfsd2a-Myc. (B) HEK293T cells were cotransfected with Mfsd2a-Myc and ZIKV E–Flag plasmids and then treated with one of the indicated pathway inhibitors (10 μM). DMSO, dimethyl sulfoxide. (C) Immunoprecipitation (IP) and immunoblot analysis of endogenous Mfsd2a with transfected ZIKV E–Flag or WNV E–Flag in HUVECs. (D) IP and immunoblot analysis of Mfsd2a-Myc with ZIKV E–Flag in HEK293T cells. (E) Coimmunoprecipitation analysis of Mfsd2a-Myc ubiquitination in HEK293T cells cotransfected with ZIKV E–Flag and ubiquitin-HA (hemagglutinin) in the presence or absence of MG132 (10 μM). (F) In vivo ubiquitination assay of endogenous Mfsd2a in mouse brain after ZIKV infection. (G) ZIKV E–mediated degradation analysis of wild-type Mfsd2a-Myc (wtM) and mutant Mfsd2a-Myc with a single point or combinational lysine mutation in HEK293T cells. Data are representative of three independent experiments with similar results.

  • Fig. 4 Uptake of TopFluor LysoPC is reduced in ZIKV-infected hBMECs.

    (A and B) TopFluor LysoPC (green) uptake assay in hBMECs that were infected with the ZIKV SZ01 strain (A) or PRVABC59 (PR59) strain (B) at the indicated MOI for 48 hours. IF staining of ZIKV E is shown in red. DAPI staining indicates the nuclei. Scale bars, 100 μm. (C and D) TopFluor LysoPC (green) uptake assay in A549 stable cells expressing Mfsd2a-Flag. Cells were infected with the ZIKV SZ01 strain (C) or PRVABC59 strain (D) at an MOI of 1 or 5 for 48 hours. IF staining of ZIKV E is shown in red. DAPI staining indicates the nuclei. Scale bars, 50 μm. (E) TopFluor LysoPC (green) uptake assay in A549 Mfsd2a stable cells that were transfected with either the ZIKV E plasmid or empty vector as the negative control (NC). Scale bar, 50 μm. (F) HEK293T cells were cotransfected with ZIKV E and either wild-type (WT) Mfsd2a or mutant Mfsd2a (K46R) plasmids for 48 hours. The cells were incubated with TopFluor LysoPC, and the fluorescence intensity was quantified.

  • Fig. 5 ZIKV infection impairs DHA uptake and lipid homeostasis.

    (A) Heat map representation of the percentage of individual phospholipid species in hBMECs. The hBMECs were infected with the ZIKV SZ01 strain at an MOI of 1 for 48 hours. Mock-infected hBMECs were used as the NC. Cells were harvested for lipidomic analysis. DHA-containing phospholipid species are highlighted in red. Each group includes three experimental repeats. Ctrl, control. (B) Lipidomic analysis of the total DHA levels and DHA-containing phospholipid species in mock- or ZIKV-infected hBMECs. The results are representative of three independent experiments. The value for each species is normalized against the total peak areas. (C and D) In vivo lipidomic analysis with ZIKV-infected neonatal mouse brains. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by one-way analysis of variance (ANOVA).

  • Fig. 6 Supplementation with DHA rescues ZIKV-induced microcephaly phenotype.

    (A) Experimental scheme of the DHA protective effect in a neonatal mouse model of ZIKV infection. The ZIKV SZ01 strain was intracerebrally (i.c.) injected into neonatal BALB/c mice at day 0 after birth. Supplementation with DHA was achieved twice via intraperitoneal (i.p.) injection at day 0 and day 3, respectively. The infected brains were inspected at day 5 and day 7. Yellow and white bars represent the brain width and brain length, respectively. Scale bars, 1 cm. (B to E) Brain width and length (B and C) and brain weight and body weight (D and E) were measured. (F to I) Brain tissues were collected at 5 or 7 dpi, and Mfsd2a and ZIKV E protein levels were analyzed by Western blotting (F and G). Viral RNA copies were analyzed by qRT-PCR (H and I). The data from three independent experiments are shown as means ± SD. Significance was calculated using a one-way ANOVA statistical test with a Dunnett’s multiple comparisons test or a two-sample Student t test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Photo credit: Jia Zhou, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaax7142/DC1

    Fig. S1. The expression of the endothelial cell marker CD31 in hBMECs.

    Fig. S2. Infection with both SZ01 and PRVABC59 only induces a slightly cytopathic effect at a high dose.

    Fig. S3. The relative protein densities of Western blot in Fig. 1.

    Fig. S4. ZIKV infection down-regulates Mfsd2a expression in JEG-3 cells.

    Fig. S5. ZIKV infection down-regulates Mfsd2a expression in vivo.

    Fig. S6. The degradation of Mfsd2a by ZIKV E can be inhibited by MG132.

    Fig. S7. Mfsd2a-overexpressing cells show a strong uptake of TopFluor-LPC.

    Table S1. Phospholipid species in mock- or ZIKV-infected hBMECs, related to Fig. 5A.

    Table S2. Phospholipid species in mock- or ZIKV-infected neonatal mouse brains, related to Fig. 5C.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. The expression of the endothelial cell marker CD31 in hBMECs.
    • Fig. S2. Infection with both SZ01 and PRVABC59 only induces a slightly cytopathic effect at a high dose.
    • Fig. S3. The relative protein densities of Western blot in Fig. 1.
    • Fig. S4. ZIKV infection down-regulates Mfsd2a expression in JEG-3 cells.
    • Fig. S5. ZIKV infection down-regulates Mfsd2a expression in vivo.
    • Fig. S6. The degradation of Mfsd2a by ZIKV E can be inhibited by MG132.
    • Fig. S7. Mfsd2a-overexpressing cells show a strong uptake of TopFluor-LPC.
    • Table S1. Phospholipid species in mock- or ZIKV-infected hBMECs, related to Fig. 5A.
    • Table S2. Phospholipid species in mock- or ZIKV-infected neonatal mouse brains, related to Fig. 5C.

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