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. Author manuscript; available in PMC: 2022 Sep 22.
Published in final edited form as: Cell Rep. 2022 Aug 16;40(7):111234. doi: 10.1016/j.celrep.2022.111234

Figure 3. eIF5A hypusination is required for KSHV LANA protein expression.

Figure 3.

(A) Schematic of LANA. LANA contains a nuclear localization sequence (NLS), proline-rich region, acidic repeat region, and DNA-binding domain. The proline-rich region (N), acidic repeat region (A), and DNA-binding domain (C) used in (F) are highlighted in boxes. (B and C) KSHV-infected TIME cells were treated with 1 mM DFMO or 5 μM GC7, with or without an addition of 20 μM spermidine.

(B and C) Whole-cell lysates were prepared at day 4 post infection and probed for anti-LANA, anti-hypusine, and anti-β-actin antibodies. (B) LANA levels were quantified using Image Lab and normalized to β-actin (biological replicates: 4; technical replicates: 2–3). (C) Extracted RNA were subjected to qPCR analysis for measuring LANA mRNA expression (biological replicates: 2; technical replicates: 2–3). ns, not significant. Error bars represent SEM.

(D) KSHV-infected TIME cells were cultured in 3D spheroid with 4% Matrigel for 2 weeks and treated with 2 mM DFMO or 10 μM GC7, with or without the supplementation of 40 μM spermidine for 5 days, followed by immunofluorescence analysis with anti-LANA antibody (red) and Hoechst 33342 (blue). Images of an entire z stack of 3D spheroid were merged and displayed. Scale bar, 10 μm. Right; graph of the average number of LANA dots per cell (biological replicates: 3).***p < 0.0005, ****p < 0.0001 using one-way ANOVA with Tukey’s multiple comparison test. Error bars represent SEM.

(E) MCF10A cells overexpressing LANA, vIRF3, and EBNA1 were treated with 0, 10, or 50 μM GC7 for 3 days. Protein expression was analyzed using anti-LANA, anti-vIRF3, anti-EBNA1, anti-hypusine, and anti-β-actin antibodies. β-actin was used as a loading control. Each representative graph of quantification of LANA, vIRF3, and EBNA1 level obtained normalized onto β-actin (biological replicates: 3–4; technical replicates: 1–2). **p = 0.0029, ***p = 0.0006; ns, not significant using one-way ANOVA with Dunnett’s multiple comparison test. Error bars represent SEM.

(F) MCF10A cells expressing FLAG-tagged N-terminal domain (N), acidic repeat region (A), or C-terminal domain (C) of LANA were treated with 0, 10, or 50 μM GC7 for 3 days. Top: amino acid sequence of each construct. Consecutive proline sequences are labeled in red. Protein expression was analyzed using anti-FLAG, anti-hypusine, and anti-β-actin antibodies. β-actin was used as a loading control. Each domain of LANA levels was quantified using Image Lab and normalized to β-actin (biological replicates: 2; technical replicates: 2–3).