Fig 1.
LMP1-deleted EBV-infected NOKs (ΔLMP1) have an enhanced Integrated Stress response compared to WT EBV- infected NOKs (WT-EBV).
(A) A Heatmap comparing the top 100 differentially expressed cellular genes between NOKs infected with WT EBV versus NOKs infected with ΔLMP1 EBV is shown. The RNAs from WT-EBV and ΔLMP1 samples were harvested in triplicates when the cells were grown in growth factor-depleted media at a sub-confluent density. Red indicates a gene is upregulated in corresponding cells and blue indicates it is down-regulated. (B) The number of genes upregulated or downregulated in ΔLMP1 EBV-infected NOKs cells compared to WT EBV-infected NOKs is shown. (C) GSEA analysis showing selected gene sets or pathways up-regulated or down-regulated in ΔLMP1 EBV-infected cells compared to WT EBV-infected cells.
Table 1.
ATF4 target genes activated in ΔLMP1 EBV-infected NOKs.
Selected genes of interest that are upregulated in ΔLMP1 EBV-infected NOKs (ΔLMP1) compared to WT EBV-infected NOKs (WT-EBV) in the RNA-seq results are shown, along with the fold-increase in gene expression and the adjusted p value.
Fig 2.
EBV inhibits the Integrated Stress Response (ISR) pathway via an LMP1-mediated effect.
(A) Uninfected NOKs (NOKs (-)), NOKs infected with EBV (WT-EBV), or NOKs infected with ΔLMP1 EBV (ΔLMP1) were plated in the absence of growth factors (EGF and BPE) in KSFM for two days at a sub-confluent density, and then harvested to perform immunoblot analysis. Expression levels of LMP1, p-eIF2α, eIF2α, ATF4, and CHOP are shown. Actin served as a loading control. (B) ΔLMP1 EBV-infected NOKs infected with a LMP1-expressing lentivirus or vector control were grown in the absence of growth factors for two days at a sub-confluent density and then immunoblot analysis was performed to examine expression levels of LMP1, p-eIF2α, eIF2α, ATF4, and CHOP as shown. Tubulin served as a loading control. (C) Comparison of expression levels of LMP1 between WT-EBV and LMP1-overexpressing ΔLMP1 EBV-infected NOKs. Tubulin served as a loading control.
Fig 3.
LMP1 inhibits the integrated stress response under a variety of different cell culture conditions and inhibits expression of the EBV immediate-early protein, BZLF1.
Uninfected NOKs (NOKs (-)), NOKs infected with EBV (WT-EBV), or NOKs infected with ΔLMP1 EBV (ΔLMP1) were plated in the presence or absence of growth factors (EGF and BPE) in KSFM for two days at a sub-confluent or confluent density, as indicated, and then harvested to perform immunoblot analysis. Expression levels of LMP1, ATF4, CHOP, Keratin-10, and the EBV immediate-early lytic protein, BZLF1, are shown. Tubulin served as a loading control.
Fig 4.
LMP1 inhibits the activity of the GCN2 and PERK kinases.
Uninfected NOKs (NOKs (-)), WT EBV-infected NOKs (WT-EBV) or ΔLMP1 EBV-infected NOKs (ΔLMP1) were grown in the absence of growth factors at a sub-confluent density for two days and immunoblot analyses were performed. (A) Expression levels of LMP1, p-GCN2, GCN2, and Actin are shown. (B) Expression levels of LMP1, p-PERK, PERK, and Actin are shown. (C) Expression levels of LMP1, p-PKR, PKR, and Actin are shown. The levels of phosphorylated protein in each condition were quantitated using image J and normalized to the level in uninfected NOKs (set as 1). The same extracts were used in the immunoblots in (B) and (C) and probed with different antibodies. The LMP1 and Actin blots used in (B) and (C) are also same.
Fig 5.
PERK and GCN2 co-operatively regulate ISR downstream targets in NOKs.
WT EBV-infected NOKs (WT-EBV) or ΔLMP1-infected NOKs (ΔLMP1), grown in the absence of growth factors at sub-confluent density for two days, were transfected with control siRNA or siRNAs targeted against PERK or GCN2, alone or in combination, as indicated and immunoblots performed. Expression levels of LMP1, PERK, GCN2, ATF4, and CHOP are shown. Tubulin served as a loading control.
Fig 6.
PERK and GCN2 co-operatively regulate spontaneous differentiation in NOKs.
WT EBV-infected NOKs (WT-EBV) or ΔLMP1-infected NOKs (ΔLMP1), grown in the absence of growth factors at sub-confluent density for two days, were transfected with control siRNA or siRNAs targeted against PERK or GCN2, alone or in combination, as indicated and immunoblots performed. Expression levels of LMP1, PERK, GCN2, and of proteins induced by differentiation of keratinocytes (IRF6, KLF4, BLIMP1, Keratin-10, Involucrin, TGM1, and SPRR1A) are shown. Tubulin served as a loading control. The extracts used in Figure 6 immunoblots are the same as those used in Fig 5. The LMP1, PERK, and GCN2 blots are the same as those used in Fig 5.
Fig 7.
GCN2 is required for TPA-induced lytic EBV reactivation and differentiation in WT EBV-infected NOKs.
WT EBV-infected NOKs cells were transfected with control siRNA or siRNAs against GCN2 as indicated, and then treated with TPA for 24 hours starting one day after siRNA transfection. (A) Immunoblot analysis was performed to examine the effect of GCN2 knock-down on proteins involved in the ISR pathway (including p-GCN2, GCN2, p-eIF2α, eiF2α, ATF4, and CHOP) and EBV lytic proteins (BZLF1, BRLF1, BMRF1, and p18 VCA). (B) Immunoblot analyses were performed to examine the effect of GCN2 knock-down on differentiation-induced cellular proteins Involucrin, BLIMP1, and TGM1. Tubulin served as a loading control for both panels. The same extracts were used for each panel, and the same GCN2, p-GCN2, and Tubulin blots were used for both panels.
Fig 8.
PERK is also required for efficient TPA-induced lytic EBV reactivation and differentiation in WT EBV-infected NOKs.
WT EBV-infected NOKs cells were transfected with control siRNA or siRNAs against PERK as indicated, and then treated with TPA for 24 hours starting one day after siRNA transfection. (A) Immunoblot analyses were performed to examine the effect of PERK knock-down on EBV lytic proteins BZLF1, BRLF1, and BMRF1. (B) Immunoblot analyses were performed to examine the effect of PERK knock-down on differentiation-induced cellular proteins BLIMP1, TGM1, Involucrin and SPRR1A. Tubulin served as a loading control for both panels. The same extracts were used for both panels, and the same PERK and Tubulin blots were used for both panels.
Fig 9.
ATF4 and CHOP are required for efficient TPA-induced lytic EBV reactivation and differentiation in WT EBV-infected NOKs.
WT EBV-infected NOKs cells were transfected with control siRNA or siRNAs against ATF4 (A and C) or CHOP (B and D) as indicated, and then treated with TPA for 24 hours starting one day after siRNA transfection. (A and B) Immunoblot analyses were performed to examine the effect of ATF4 and CHOP knock-down on lytic EBV proteins BZLF1, BRLF1, BMRF1 and p18 VCA as indicated. (C and D) Immunoblot analyses were performed to examine the effect of ATF4 and CHOP knock-down on TPA-induced differentiation markers BLIMP1, TGM1, and SPRR1A as indicated. Tubulin served as a loading control. The cellular extracts used in (A) and (C) or (B) and (D) were the same. The same ATF4 and Tubulin blots were used in (A) and (C), and the same CHOP and Tubulin blots were used for (B) and (D).
Fig 10.
CHOP overexpression induces lytic reactivation and differentiation in NOKs and NPC43 cells but not in Mutu I Burkitt B cells.
WT EBV-infected NOKs (WT-EBV), EBV infected nasopharyngeal carcinoma NPC43 cells, or EBV-infected Mutu I Burkitt lymphoma cells were stably infected with a doxycycline inducible CHOP expressing or control lentiviruses and puromycin selected to obtain stable cell lines. WT-EBV or NPC 43 cells were then treated with 500ng/ml doxycycline, and Mutu I cells were treated with 1000 ng/ml doxycycline for 48 hours and harvested to perform immunoblot analysis. (A) Expression of CHOP, EBV lytic markers BZLF1, BRLF1, BMRF1, p18 VCA, and Tubulin in EBV-infected NOKs. (B) Expression of CHOP, differentiation markers IRF6, BLIMP1, KLF4, ELF3, Keratin-10, Involucrin, TGM1, SPRR1A, and Tubulin in EBV-infected NOKs. (C) Expression of CHOP, EBV lytic proteins BZLF1, BRLF1, BMRF1,p18 VCA, and Tubulin in EBV- infected NPC43 cells. (D) Expression of CHOP, differentiation markers IRF6, BLIMP1, KLF4, ELF3, Keratin-10, Involucrin, TGM1, SPRR1A, and Tubulin in EBV-infected NPC43 cells. (E) Comparison of expression of EBV lytic markers BZLF1, BRLF1, and BMRF1 in EBV-infected Mutu I cells and NOKs when similar levels of CHOP expression were induced by doxycycline treatment. Tubulin serves as a loading control. (F) Comparison of expression of epithelial cell differentiation markers IRF6, BLIMP1, KLF4, ELF3, Keratin-10, Involucrin, TGM1, SPRR1A in EBV-infected Mutu I cells and EBV-Infected NOKs when similar levels of CHOP expression were induced. Tubulin served as a loading control.The cellular extract used to probe the blots in (A) and (B) was the same, the cellular extract used to probe the blots in (C) and (D) was the same, and the cellular extract used to probe the blots in (E) and (F) was the same. The same CHOP and Tubulin blots were used in (A) and (B), the same CHOP and Tubulin blots were used in (C) and (D), and the same Tubulin blot was used in (E) and (F).
Fig 11.
CHOP induces lytic reactivation in EBV-infected NOKs cells through transcription factors KLF4 and BLIMP1.
WT EBV-infected NOKs (WT-EBV) stably infected with a doxycycline inducible CHOP-expressing or control lentivirus were transfected with control siRNA or siRNAs directed to KLF4 or BLIMP1 as indicated. 24h following transfection, cells were treated with 500ng/ml doxycycline for 48 hours and immunoblot analyses were performed to examine expression of BLIMP1, KLF4, BZLF1, BRLF1, BMRF1, and p18 VCA as shown. Tubulin served as a loading control.
Fig 12.
Low level and high level LMP1 expression have different effects on CHOP and BZLF1 expression in ΔLMP1 EBV-infected NOKs.
ΔLMP1 EBV-infected NOKs (ΔLMP1) were stably infected with a lentivirus expressing a doxycycline inducible LMP1 gene, treated for two days with various doses of doxycycline, and then immunoblot analyses were performed to examine expression of LMP1, CHOP and BZLF1 as indicated. The levels of LMP1 expressed in untreated or TPA-treated EBV WT NOKs (WT-EBV), or in three different EBV-transformed lymphoblastoid cell lines (infected with the Mutu I (LCL-1), AG876 (LCL-2) and Akata (LCL-3) strain viruses) are shown in the left panel, and the levels of CHOP and BZLF1 are shown on the right panel. The levels of CHOP and BZLF1 were quantitated using Image J and the level in untreated cells was set as 1. Tubulin served as a loading control.
Fig 13.
A model for how LMP1 regulates lytic EBV reactivation and epithelial cell differentiation through inhibition of the ISR pathway.
See text for details of the model. The DNA symbol used in this figure was “Created with BioRender.com”.