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. 2023 Oct 20;9(42):eadj4198.
doi: 10.1126/sciadv.adj4198. Epub 2023 Oct 20.

Hepatoviruses promote very-long-chain fatty acid and sphingolipid synthesis for viral RNA replication and quasi-enveloped virus release

Tomoyuki Shiota  1 , Zhucui Li  2 , Guan-Yuan Chen  2 , Kevin L McKnight  1 , Takayoshi Shirasaki  1 , Bryan Yonish  1 , Heyjeong Kim  3 , Ethan J Fritch  3 , Timothy P Sheahan  4 , Masamichi Muramatsu  5 , Maryna Kapustina  6 , Craig E Cameron  1   3 , You Li  1   7 , Qibin Zhang  2   8 , Stanley M Lemon  1   3   7
Affiliations

Hepatoviruses promote very-long-chain fatty acid and sphingolipid synthesis for viral RNA replication and quasi-enveloped virus release

Tomoyuki Shiota et al. Sci Adv. .

Abstract

Virus-induced changes in host lipid metabolism are an important but poorly understood aspect of viral pathogenesis. By combining nontargeted lipidomics analyses of infected cells and purified extracellular quasi-enveloped virions with high-throughput RNA sequencing and genetic depletion studies, we show that hepatitis A virus, an hepatotropic picornavirus, broadly manipulates the host cell lipid environment, enhancing synthesis of ceramides and other sphingolipids and transcriptionally activating acyl-coenzyme A synthetases and fatty acid elongases to import and activate long-chain fatty acids for entry into the fatty acid elongation cycle. Phospholipids with very-long-chain acyl tails (>C22) are essential for genome replication, whereas increases in sphingolipids support assembly and release of quasi-enveloped virions wrapped in membranes highly enriched for sphingomyelin and very-long-chain ceramides. Our data provide insight into how a pathogenic virus alters lipid flux in infected hepatocytes and demonstrate a distinction between lipid species required for viral RNA synthesis versus nonlytic quasi-enveloped virus release.

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Figures

Fig. 1.
Fig. 1.. Quantitative lipidomics analysis of HAV p16-infected hepatoma cells.
(A) Relative molar abundance (mol %) of major lipid classes 14 days in (top) mock-infected or (bottom) p16 virus–infected Huh-7.5 cells. SM, sphingomyelin. (B) Major lipid classes in triplicate cell samples collected 7 and 14 days after mock or p16 virus infection, rank ordered by relative abundance in uninfected cells on day 7: LPC, lysophosphatidylcholine; PG, phosphatidylglycerol; PI, phosphatidylinositol; LPE, lysophosphatidylethanolamine; PC-O, ether-linked phosphatidylcholine; SPB, sphingosine; PE-O, ether-linked phosphatidylethanolamine; LPC-O, ether-linked lysophosphatidylcholine. *50% increase or decrease from mock at day 14, with Q < 0.01. (C) Heatmap of major lipid classes, with clustering based on Spearman rank order correlation (65). (D) Heatmap showing molar abundance of different CE species grouped by ascending total carbon number. (E) Fold change in percent molar abundance of lipids with very-long-chain fatty acid (VLCFA) tails >22 carbons within each lipid class at 7 and 14 dpi. Data shown are means ± SD. (F) Fold change in molar abundance of CE, PC, DG, and TG with VLCFA tails 14 dpi plotted as a function of the total number of carbon atoms in acyl tails. *P < 0.05, ***P < 0.001, ****P < 0.0001 by Spearman correlation. (G) Heatmap of lipid species with >3 double bonds (PUFA) with lipid class grouped by Spearman rank correlation. (H) Relative molar abundance of PUFA by lipid class. *Q < 0.01 at both 7 and 14 dpi. (I) Relative molar abundance of lipids with odd-number carbon fatty acid (OCFA) tails in mock- and p16-infected cells at 7 and 14 dpi. Q < 0.01 at both 7 and 14 dpi. (J) Percent molar abundance of lipids containing OCFA tails with defined numbers of carbon atoms.
Fig. 2.
Fig. 2.. VLCFA synthesis is required for hepatovirus replication.
(A) VLCFA synthesis pathway. (B) Immunoblots of ACC1 in lysates of ACC1-KO92 and control (sgCtrl) cells. (C) Maximum intensity projection confocal images of clonal ACC1-KO89.1 cells with targeted CRISPR deletion of ACACA and parental Huh-7.5 cells showing lipid droplets stained with BODIPY (green) and CellMask membrane dye (red). HexCer, hexosylceramide. (D) Top: 18f-NLuc HAV reporter virus genome. Bottom: NLuc expressed by 18f-NLuc virus in clonal ACC1-depleted cell lines, ACC1-KO89.1 and ACC1-KO92.5, 72 hours after infection. (E) Immunoblot showing 17β-HSD12 expression in cells with targeted CRISPR deletion of HSD17B12 (encoding 17β-HSD12) and sgCtrl cells. (F) Maximum intensity projection confocal image of HSD12-KO54.10 cells stained with BODIPY-C9. (G and H) NLuc expression in 18f-NLuc–infected HSD17-KO54.10 (G) or HSD17-KO51.T3 (H) versus control sgCtrl cells. ****P < 0.0001. (I) Fold increase in NLuc expressed by 18f-NLuc virus 72 hours after infection of ACC1-depleted, 17β-HSD12–depleted, or sgCtrl cells supplemented with fatty acids. (J) Top: Subgenomic 18f-FLuc replicon RNA; GAA mutation ablates replication. Bottom: FLuc expressed by HSD12-KO54.10 and sgCtrl cells transfected with 18f-FLuc RNA or sgCtrl cells transfected with 18f-FLuc/GAA. ****P < 0.0001. (K) Relative molar abundance of lipids with VLCFA tails in HSD12-KO54.10 and sgCtrl cells. (L) Class distribution of lipids with VLCFA tails in sgCtrl and 17β-HSD12–depleted cells. Pie chart area is proportional to molar abundance. (M) Fold change in relative molar abundance of CE, PC, and TG with VLCFA tails plotted against numbers of carbon atoms in acyl tails. s = Spearman correlation (P < 0.0001 for each class). (N) Relative molar abundance of lipids with odd-number carbon fatty acid (OCFA) tails in sgCtrl and 17β-HSD12–depleted cells. Q < 0.05 for each lipid class shown. (O) Relative molar abundance of polyunsaturated lipids (>3 double bonds) in sgCtrl and 17β-HSD12–depleted cells. Q < 0.05 for each class shown.
Fig. 3.
Fig. 3.. Lipid composition of extracellular eHAV and eHAV-producer cells.
(A) Isopycnic gradient profile showing fractions containing eHAV selected for LC-MS/MS lipidomics analysis. GE = HAV genome equivalents. (B) Normalized percent molar abundance (mol %) of major nonglycerolipid lipid classes in eHAV-producer cells (left), eHAV (center) and control EV (right) samples. (C) Mean mol % abundance of individual species of major lipid classes identified in eHAV-producer cells, eHAV, or EV, ±SD, n = 3. SM(d34.1) abundance in eHAV and EVs is highlighted. (D) Enrichment of major lipid classes in eHAV versus HAV-infected producer cell samples. *Q < 0.01 by t test with two-stage step-up Benjamini, Krieger, and Yekutieli test. (E) Heatmap showing relative abundance of individual nonhexosyl Cer species identified in both eHAV and producer cells (F) Top: Normalized mean percent distribution of Cer species by carbon number in eHAV and producer cell samples (values shown as percent of PC). Bottom: Correlation between the fold change in relative molar abundance of individual PC species in eHAV versus producer cells, and total carbon number. r = Spearman correlation coefficient. P = 0.091. Similar analyses are shown for: (G) SM, P = 0.042; and (H) PC, P = <0.0001. (I) Mean normalized mol % of PUFA (≧4 double bonds) by lipid class in producer cells versus eHAV. Cholesterol esters excluded from producer cell data for comparability with eHAV. *Q < 0.01. (J) Means normalized mol % of lipids with odd-number carbon fatty acids (OCFAs) by lipid class in producer cells versus eHAV. *Q < 0.01; “Other” includes PS and PI which were also Q < 0.01.
Fig. 4.
Fig. 4.. Transcriptomic analysis of p16 virus-infected cells.
(A and B) Volcano plots of differentially expressed transcripts with >10 reads, fold change (FC) >2 and adjusted P (padj) <0.01 at (A) 7 (n = 1130, 823 up-regulated and 307 down-regulated) and (B) 14 dpi (n = 1553, 950 up-regulated and 603 down-regulated). (C). Normalized enrichment score (NES) of hallmark gene sets with Q < 0.25. (D) Infection-related changes in selected transcripts. Left: Bars represent transcripts per million (TPM) at14 dpi. Right: Fold change from mock-infected cells 7 and 14 dpi. Shading indicates Q < 0.01. (E) Immunoblots (IB) of ELOVL4 and ELOVL7. Mean fold increase of ELOVL4 relative to β-actin at 7 and 14 dpi was 4.0 ± 2.24 SEM, n = 6 blots, P = 0.03 (Wilcoxon sign rank test); ELOVL7, 1.5-fold ±0.37 SEM (P = 0.38, n = 4). (F) Immunoblots of ELOVL4 and ELOVL7 in cells transduced with nontargeting or ELOVL4-specific short hairpin RNA (shRNA), and transfected with nontargeting or ELOVL7-specific siRNA. (G) Left: NLuc expressed by cells shown in (F) 48 hours after infection with quasi-enveloped 18f-NLuc virus. Data from technical replicates in one of four representative experiments. Right: Cell viability assessed by ATP assay. (H) Long-chain acyl-CoA synthetase family transcript (left) abundance 14 dpi and (right) fold change from mock-infected cells 7 and 14 dpi. (I) Immunoblots of ACSL5 at 7 and 14 dpi. (J) Three-dimensional volume reconstruction of Airyscan fluorescence images of mock- or p16 virus–infected cells following 60 min incubation with BODIPY-C16 (1 μg/ml). (K) Mean cytoplasmic voxel BODIPY-C16 fluorescence intensity in mock (n = 271) versus infected cells (n = 328). P value by two-sided Mann-Whitney test. Data from two independent experiments. au, arbitrary units. (L) Immunoblots of ACSL5 in cells transfected with ACSL5-specific versus nontargeting (siCtrl) siRNA, ±0.5 mM oleic acid (OA, cis-9-octadecenoic acid, 18:1) supplementation. (M) NLuc expressed by 18f-NLuc virus 72 hours postinfection. Data are technical replicates from one of four independent experiments with similar results.
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