Fig 2: ODC1 is required for KSHV lytic reactivation.(A, B). ODC1 knockdown in HEK293.r219 cells transfected with ODC1 siRNAs (si1, si2, si3) or non-targeting control siRNA (NT) was analyzed by RT-qPCR (A) or immunoblotting (B). (C, D). Intracellular polyamine species (Put, Spd, Spm) in HEK293.r219 cells transfected with ODC1 siRNAs (si1, si2, si3) or NT were analyzed by TLC with pure individual polyamine species used as reference (C). Relative polyamine amount (Put, Spd, Spm) in above cells were quantified (average of ODC1 si1-3) and normalized to NT-transfected cells (D). Results were calculated from n = 2 independent repeats. (E-G). HEK293.r219 cells transfected with ODC1 siRNAs (si1, si2, si3) or NT were treated with TPA (20 ng/mL) + NaB (0.3mM) for 48h, and visualized by fluorescence imaging (E). Expression of RFP protein indicates KSHV lytic reactivation, while GFP signal means that cells are KSHV-infected. mRNA level of KSHV lytic genes (ORF50/RTA, K8/K-bZIP, ORF26) in above cells was analyzed by RT-qPCR assays and normalized to NT-transfected induced cells (F). Protein level of KSHV lytic genes (ORF45, K8.1A/B) was analyzed by immunoblotting assays (G). GAPDH was used as the loading control. Results were calculated from n = 3 independent experiments and presented as mean ± SEM (*** p<0.001, 1-way ANOVA (Tukey test) and 2-way ANOVA (Bonferroni test)).

Fig 3: Inhibition of eIF5A hypusination efficiently blocks KSHV lytic infection.(A). mRNA level of KSHV lytic genes (ORF50/RTA, K8/K-bZIP and ORF26) in iSLK.BAC16 cells pre-treated with progressively increasing doses of GC7 for 24h and subsequently induced with Dox (1µg/mL) for 48h were analyzed by RT-qPCR assays and normalized to mock-treated induced cells. The drug effect at a series of doses was plotted as percentage of maximum response by using GraphPad PRISM 5. IC50 was determined for GC7’s inhibitory effect. (B). Protein level of KSHV ORF45 and hyp-eIF5A in iSLK.BAC16 cells pre-treated with GC7 (12.5µM) for 24h and subsequently induced with Dox (1µg/mL) up to 48h was analyzed by immunoblotting assays. GAPDH was used as the loading control. (C-E). mRNA level of KSHV lytic genes (PAN RNA, K8.1, ORF26) in TREx BCBL1-RTA cells pre-treated with progressively increasing doses of GC7 for 24h and subsequently induced with Dox (1µg/mL) for 48h were analyzed by RT-qPCR assays and normalized to mock-treated induced cells. The drug effect at a series of doses was plotted as percentage of maximum response by using GraphPad PRISM 5 (C). IC50 was determined for GC7’s inhibitory effect. Protein level of KSHV lytic genes (ORF45, K8.1A/B, ORF26) in above cells was analyzed by immunoblotting assays (D). GAPDH was used as the loading control. Relative copy number of KSHV viral genomes was also determined (E). (F). mRNA level of KSHV lytic genes (ORF50/RTA, PAN, K8, K8/K-bZIP, ORF26) in HEK293.r219 cells pre-treated with GC7 for 24h and subsequently induced with TPA (20 ng/mL) + NaB (0.3mM) for 48h was analyzed by RT-qPCR assays and normalized to mock-treated induced cells. (G). mRNA level of KSHV lytic genes (K8/K-bZIP and ORF26) in TIME.BAC16 cells pre-treated with GC7 for 24h and subsequently induced with TPA (20 ng/mL) + NaB (0.5mM) for 48h was analyzed by RT-qPCR assays and normalized to mock-treated induced cells. (H, I). TIME cells were de novo infected with KSHV BAC16 viruses (MOI = 1). Unbound viruses were washed away, and cells were subsequently treated with increasing doses of GC7 for 48h, then subjected to fluorescence imaging analysis (H). Nuclei were labelled with Hoechst (Blue), while GFP expression indicated infection with KSHV BAC16 viruses. MFI of GFP expression from nine different fields of view for =105 cells was measured and normalized to mock-treated infected cells (I). (J, K). Primary tonsillar B cells isolated from 4 healthy donors were de novo infected with KSHV BAC16 viruses (MOI = 3). Unbound viruses were washed away, and cells were subsequently treated with GC7 (12.5 µM) for 72h, followed by RT-qPCR assays to measure mRNA level of KSHV lytic genes (ORF50/RTA, K8/K-bZIP) and latent gene (ORF73/LANA) and normalized to mock-treated cells (J). Cell viability was analyzed by CellTiter-Glo assays and normalized to mock-treated cells (K). Results were calculated from n = 3–4 independent experiments and presented as mean ± SEM (* p<0.05; *** p<0.001, two-tailed paired Student t-test, 1-way ANOVA (Tukey test) and 2-way ANOVA (Bonferroni test)).

Fig 4: Polyamine depletion efficiently blocks KSHV lytic reactivation.(A, B). HEK293.r219 cells pretreated with progressively increasing doses of 2-difluoromethylornithine (DFMO) for 24h were subsequently induced by ectopic expression of ORF50 for 48h or left un-induced using the empty vector. Above cells were then visualized by fluorescence imaging (A). mRNA level of KSHV K8/K-bZIP (left panel) and latent genes (ORF71/v-FLIP, ORF72/v-Cyclin, and ORF73/LANA; right panel) in above cells were analyzed by RT-qPCR assays and normalized to mock-treated induced cells. The drug effect at a series of doses was plotted as percentage of maximum response by using GraphPad PRISM 5. Relative copy number of KSHV genomes (middle panel) was also analyzed. IC50 was determined for DFMO’s inhibitory effect. (C). Exogenous polyamines (mixed polyamines supplement [PA Supp, 5x], Spermidine [Spd, 10µM] or Spermine [Spm, 10µM]) were complemented into the culture media of HEK293.r219 cells treated with DFMO (500µM) and induced by ectopic expression of ORF50 for 48h or left un-induced using the empty vector. The above cells were labelled with Hoechst and visualized by fluorescence imaging to determine percentage of RFP-positive cells (left panel). mRNA level of KSHV lytic genes (K8/K-bZIP [middle panel], ORF26 [right panel]) in above cells were analyzed by RT-qPCR assays. All results were normalized to mock-treated ORF50-induced cells. (D, E). mRNA level of KSHV lytic genes (ORF50/RTA, K8/K-bZIP and ORF26) in iSLK.BAC16 cells pre-treated with increasing doses of DFMO for 24h and subsequently induced by Dox (1µg/mL) + NaB (1mM) for 48h were analyzed by RT-qPCR assays and normalized to mock-treated induced cells (D, left panel). Relative copy number of KSHV genomes in above cells was also analyzed (D, right panel). Protein level of KSHV lytic genes (ORF45, K8.1A/B) was analyzed by immunoblotting (E). GAPDH was used as the loading control. (F). TIME cells were de novo infected with KSHV BAC16 viruses (MOI = 1). Unbound viruses were washed away, and cells were subsequently treated with increasing doses of DFMO for 48h with or without addition of polyamines supplement (PA Supp, 5x) to the culture media. Cells were then subjected to fluorescence imaging analysis (top panel). Nuclei were labelled with Hoechst and used to normalized GFP fluorescence. MFI of GFP expression from nine different fields of view for =105 cells was measured and normalized to mock-treated cells (bottom panel). (G). TIME.BAC16 cells were pre-treated with DFMO for 24h with or without addition of polyamines supplement (PA Supp, 5x) to the culture media. Cells were subsequently induced with TPA (20 ng/mL) + NaB (0.5mM) for 48h, and mRNA level of KSHV lytic genes (K8/K-bZIP and ORF26) were analyzed by RT-qPCR assays and normalized to mock-treated, induced cells. Results were calculated from n = 3–4 independent experiments and presented as mean ± SEM (* p<0.05; ** p<0.01; *** p<0.001, 1-way ANOVA (Tukey test) and 2-way ANOVA (Bonferroni test)).

Fig 5: eIF5A hypusination is regulated by KSHV and required for its lytic reactivation.(A). Schematic view of spermidine consumption for eIF5A hypusination. (B). mRNA level of DHPS during KSHV lytic reactivation in TREx BCBL1-RTA (mock or Dox-treated, 48h) was analyzed by qPCR assay and compared to KSHV-negative BJAB cells under the same conditions. (C, D). mRNA (C) and protein (D) levels of DHPS in KSHV-infected PEL cells lines (BCBL1 [wild-type], TREx BCBL1-RTA, BC-3) and KSHV-negative BJAB cells were analyzed by qPCR and protein immunoblotting assays, respectively. (E). Protein level of total eIF5A and hyp-eIF5A in KSHV-infected PEL cells lines described in (D) was measured by protein immunoblotting. (F). Protein level of total eIF5A (top) or hyp-eIF5A (bottom) in TREx-BCBL1-RTA cells treated with Dox or mock were measured by dual-color immunostaining (Alexa fluor 488 for total eIF5A, and Alexa Fluor 647 for hyp-eIF5A) and flow cytometry at 24 and 48hpi. (G, H). DHPS knockdown and hyp-eIF5A protein level in iSLK.BAC16 cells transfected with DHPS siRNAs (si1, si2) or NT was analyzed by protein immunoblotting (G). mRNA level of KSHV lytic genes (ORF50/RTA, K8/K-bZIP and ORF26) in above cells induced with Dox (1μg/mL) for 48h was analyzed by RT-qPCR assays and normalized to NT-transfected, induced cells (H). (I). Effect of ODC1 knockdown on hyp-eIF5A protein level in iSLK.BAC16 cells transfected with ODC1 siRNAs (si1, si2, si3) or NT was analyzed by protein immunoblotting. Intensity of protein bands was determined by using AlphaView SA (software) and normalized to the loading control GAPDH. Results were calculated from n = 3–4 independent experiments and presented as mean ± SEM (* p<0.05; ** p<0.01; *** p<0.001, 1-way ANOVA (Tukey test) and 2-way ANOVA (Bonferroni test)).