Journal: bioRxiv
Article Title: Epstein-Barr Virus Latent Membrane Protein 1 Suppresses Ferroptosis via Pentose Phosphate Pathway and Glutathione Metabolism
doi: 10.64898/2026.03.09.710628
Figure Lengend Snippet: (A) Volcano plot analysis of host transcriptome-wide genes differentially expressed in primary B cells from three independent donors infected with WT or TES2m LMP1 for 21 days. Positive fold changes indicate higher transcripts in cells with TES2m LMP1. (B) PFKFB4 glucose metabolism roles. Glucose is metabolized by glycolysis or by the PPP. Glycolysis flux is regulated via Fructose-2,6-BP, an allosteric regulator of PFK1. PFKFB3 phosphorylates Fructose-6-P into Fructose-2,6-BP while PFKFB4 phosphatase activity converts Fructose-2,6-BP to Fructose-6-P to shunt glycolytic flux to PPP. Created in BioRender. Burton, E. (2026) https://BioRender.com/i0gawc8 . (C) KEGG pathway analysis of transcripts significantly upregulated in cells infected by EBV with WT LMP1 at day 21 post infection. (D) KEGG pathway analysis of transcripts significantly upregulated in cells infected by TES2m EBV at day 21 post infection. (E) PFKFB4 protein levels in WT versus TES2m LMP1 EBV infected cells. Cells from two independent donors were infected with EBV with WT LMP1 or TES2m LMP1 for 22 days. Densitometry values of PFKFB4 vs β-Actin load controls are shown. (F) Effects of LCL LMP1 KO on PFKFB4 mRNA. Median PFKFB4 reads from RNAseq analysis of Cas9+ GM12878 cells with control vs LMP1 targeting sgRNA expression for 48 hours. Shown are data from n=3 RNAseq datasets. (G) PFKFB4 protein levels in LMP1 KO LCLs. Cas9+ GM12878 were mock induced or induced to express LMP1 sgRNA for 48 hours. WCL was extracted and immunoblot was carried out using the indicated antibodies. (H) Effects of TES1 vs TES2 signaling inhibition on LCL PFKFB4 expression. Median PFKFB4 RNAseq reads from RNAseq analysis of Cas9+ GM12878 expressing LMP1 sgRNA together with dox-induced LMP1 TES1m vs WT LMP1 cDNA expression. Shown are data from n=3 RNAseq datasets. (I) Relative PFKFB4 protein abundances from proteomic analysis of primary human B-cells at the indicated days post-infection by the EBV B95.8 strain. Shown are mean ± SD values from n=4 replicates. (J) Effects of exogenous PFKFB4 expression on proliferation of primary B cells infected by EBV with WT versus TES2m LMP1. Peripheral blood B cells from three independent donors were infected with EBV with WT vs TES2m LMP1. After 14 days, cells were transduced with lentivirus control or with lentivirus driving stable PFKFB4 expression. Five days post-selection, cells were re-seeded. Twelve days later, live cell counts were defined. P-values were determined by one-sided Fisher’s exact test. * p<0.05, **p<0.005, ***p<0.0005. blots are representative of n=3 independent replicates.
Article Snippet: To construct indexed libraries, 1 μg of total RNA was used for polyA mRNA selection using NEBNext Poly(A) mRNA Magnetic Isolation Module (Cat#E7490S), and library preparation with NEBNext Ultra RNA Library Prep with Sample Purification Beads (Cat#E7765S).
Techniques: Infection, Activity Assay, RNA sequencing, Control, Expressing, Western Blot, Inhibition, Transduction, Selection
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