Chikungunya Replication and Infection Is Dependent upon and Alters Cellular Hexosylceramide Levels in Vero Cells

Abstract

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes significant global morbidity, including fever, rash, and persistent arthralgia. Utilizing untargeted lipidomics, we investigated how CHIKV infection alters host cell lipid metabolism in Vero cells. CHIKV infection induced marked catabolism of hexosylceramides, reducing their levels while increasing ceramide byproducts. Functional studies revealed a reliance on fatty acid synthesis, β-oxidation, and glycosphingolipid biosynthesis. Notably, inhibition of uridine diphosphate glycosyltransferase 8 (UGT8), essential for galactosylceramide production, significantly impaired CHIKV replication and entry in Vero cells. Sensitivity of CHIKV to UGT8 inhibition was reproduced in a disease-relevant cell line, mouse hepatocytes (Hepa1-6). CHIKV was also sensitive to evacetrapib, a cholesterol ester transfer protein (CETP) inhibitor, though the mechanism of inhibition appeared independent of CETP itself, suggesting an off-target effect. These findings highlight specific lipid pathways, particularly glycosphingolipid metabolism, as critical for CHIKV replication and further refine our understanding of how CHIKV exploits host lipid networks. This study provides new insights into CHIKV biology and suggests that targeted investigation of host lipid pathways may inform future therapeutic strategies.

Keywords: alphavirus; chikungunya virus; fatty acid synthesis; hexosylceramide; lipidomics.

Figure 1

CHIKV infection decreases HexCer levels. (A) Schematic of the lipidomics experiment. Created in biorender.com. (B) PCA analysis of the lipidomic samples in both the positive and negative ion modes. (C) Fold change in levels of lipids found in chikungunya (CHIKV) and vesicular stomatitis virus (VSV) infected cells compared to uninfected cells. (D–F) Relative abundances of lipids driving the PCA analysis. Ceramide (Cer), hexosylceramides (HexCer), phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylserine (PS). Two-way ANOVA with post hoc Tukey’s multiple comparisons test. *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001, ****, p ≤ 0.0001.

Figure 2

CHIKV infection-associated cellular lipidome. (A) Volcano plots of untargeted mass-spec lipidomic datasets in CHIKV-infected cells compared to uninfected controls in both positive (Left) and negative (Right) ion modes. (B–G) Bar charts of lipid species identified via multivariate analysis to contribute to significant variance between CHIKV-infected and mock-infected lipidomes. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), diacylglycerol (DG), phosphatidylglycerol (PG), hexosylceramide (HexCer), ceramide (Cer). Data are based on the averages of n = 5 replicates. For (A), significance was determined via multiple unpaired t-tests (Benjamini, Krieger, Yekutieli, FDR = 1.0%). In (B–G), Two-way ANOVA with post hoc Sidak’s multiple comparisons test was employed for univariate analysis. *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001, ****, p ≤ 0.0001.

Figure 3

Altering lipid regulatory pathways reduced CHIKV replication. (A) CHIKV virus contains both a GFP and nano-luciferase (NLuc) to readily monitor virus replication. (B) Pathways that can lead to transcriptional regulation of lipid regulatory pathways. (C) Relative CHIKV replication and cell viability with MK2206, rosiglitazone, and GW3965. Data represent the average of triplicates completed in three independent trials compared to the DMSO control. An unequal variance (Welch’s correction) t-test was performed for normalized data. *, p < 0.05; **, p < 0.01. (A,B) created with biorender.com.

Figure 4

CHIKV replication is sensitive to disruption of cellular fatty acid synthesis. (A) Pathway describing fatty acid synthesis, activation, and catabolism, created in biorender.com. (B) CHIKV replication (dark grey) and cell viability (pink) when infection occurs in the presence of the indicated inhibitor. Data represent the average of triplicates completed in at least three independent trials compared to the DMSO control. An unequal variance (Welch’s correction) t-test was performed for normalized data. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 5

CHIKV replication is sensitive to lipogenesis and lipolysis inhibitors. (A) Phospholipid to triacylglyceride anabolic and catabolic pathway, with complex phospholipid biosynthesis branches, created in biorender.com. Lyso-phosphatidic acid (Lyso-PA), monoacyglycerol (MAG), phosphatidylinositol (PI), cardiolipin (CL) (B) CHIKV replication (dark grey), and cell viability (pink) when infection occurs in the presence of the indicated inhibitor. Data represent the average of triplicates completed in at least three independent trials compared to the DMSO control. An unequal variance (Welch’s correction) t-test was performed for normalized data. *, p < 0.05; **, p < 0.01.

Figure 6

CHIKV replication and ceramide synthesis. (A) Ceramide and sphingolipid synthesis pathway, created in biorender.com. (B) CHIKV replication (dark grey) and cell viability (pink) when infection occurs in the presence of the indicated inhibitor. Data represent the average of triplicates completed in at least three independent trials compared to the DMSO control. An unequal variance (Welch’s correction) t-test was performed for normalized data. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 7

CHIKV and cholesterol. (A) Cholesterol uptake pathway and (B) cholesterol synthesis pathways; created in biorender.com. (C) CHIKV replication (dark grey) and cell viability (pink) when infection occurs in the presence of the indicated inhibitor. Data represent the average of triplicates completed in at least three independent trials compared to the DMSO control. An unequal variance (Welch’s correction) t-test was performed for normalized data. *, p < 0.05; **, p < 0.01.

Figure 8

CETP inhibitors limit CHIKV replication. (A) Schematic of CETP function; created in biorender.com. (B) CHIKV replication (dark grey) and cell viability (pink) when infection occurs in the presence of the indicated inhibitor. Data represent the average of triplicates completed in at least three independent trials compared to the DMSO control. An unequal variance (Welch’s correction) t-test was performed for normalized data. *, p < 0.05; **, p < 0.01.

Figure 9

Mechanism of inhibition. (A) CHIKV spread over 24 h, MOI = 0.001. (B) Supernatant titers after 24 h treatment, MOI = 0.001. (C) Virucidal assay. Viral stocks were incubated with compounds for 1 h, then titrated. (D) Virus entry after short, 1 h compound pre-treatment. (E) Virus entry after long, 18 h compound pre-treatment. (F) CHIKV replicon assay. When listed, compounds were at the following concentrations: DMSO [0.1% v/v], evacetrapib [15 µM], D609 [100 µM], UGT8-In19 [25 µM], CAY10499 [50 µM], NH₄Cl [10 mM], 2-BP [50 µM]. Data represent the mean ± SEM from at least three independent trials. Student t-test was used to compare treated samples with DMSO control. An unequal variance (Welch’s correction) t-test was performed for normalized data. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 10

UGT8-In19 inhibits CHIKV in Hepa1-6 cells. (A) CHIKV spread occurred over 48 h in Hepa1-6 cells, MOI = 0.001. Cell viability of uninfected UGT8-In19 [8.3 µM] treated cells is shown compared to DMSO control. (B) Supernatant titers after 48 h treatment, MOI = 0.01. Data represent the mean ± SEM from at least three independent trials. Student t-test was used to compare treated samples with DMSO control. An unequal variance (Welch’s correction) t-test was performed for normalized data. *, p < 0.05; **, p < 0.01.

Figure 11

Evacetrapib inhibition is independent of serum lipoproteins. VeroS cells were infected with CHIKV (MOI = 0.001) and treated with Evacetrapib or DMSO within DMEM-media containing normal FBS or lipoprotein-depleted FBS (LD-FBS); cells were harvested at 24 h and percent infection determined via flow cytometry. Two-way ANOVA with post hoc Sidak’s multiple comparison test was employed for statistical analysis. **, p ≤ 0.01, ***, p ≤ 0.001.