1. B Cell Receptor Activation and Chemical Induction Trigger Caspase-Mediated Cleavage of PIAS1 to Facilitate Epstein-Barr Virus Reactivation 
Kun Zhang, Dong-Wen Lv, Renfeng Li 
Cell Reports 
Volume 21, Issue 12, Pages (December 2017)
DOI: /j.celrep
Copyright © 2017 The Author(s) Terms and Conditions

2. Cell Reports 2017 21, 3445-3457DOI: (10.1016/j.celrep.2017.11.071)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1 PIAS1 Depletion Facilitates EBV Lytic Replication
(A) Schematic representation of Cas9 target sites for guide RNAs sg-1 and sg-2. ORF, open reading frame.
(B) Akata (EBV+) cells were used to establish stable cell lines using guide RNA constructs sg-1 and sg-2 and a non-targeting control (NC). Western blot analyses show PIAS1 depletion in the two cell lines as indicated.
(C) PIAS1 depletion leads to enhanced lytic gene expression. PIAS1-depleted (sg-1) and control cells were either untreated or treated with anti-IgG for 24 hr. RNAs from these cells were extracted and analyzed by qRT-PCR, using primers as indicated. The values of control without treatment were set as 1.
(D and E) PIAS1 depletion leads to enhanced EBV protein expression and viral DNA replication. The PIAS1-depleted and control cells were treated with anti-IgG-induced cross-linking of BCR. Viral protein expression was monitored by western blot (WB) using antibodies as indicated in (D), and viral DNA replication was measured by qPCR in (E). The value of control at 0 hr (lane 3) was set as 1.
In (C) and (E), representative results from three biological replicates are presented. Error bars indicate SD. ∗p < 0.01; ∗∗p <
See also Figure S1.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

4. Figure 2 PIAS1 Reconstitution Suppresses EBV Lytic Replication
(A) Akata (EBV+) sg-1 cells were used to establish PIAS1-expressing stable cell lines using a pLX-PIAS1 lentiviral construct. Western blot analyses show PIAS1, ZTA, RTA, and BGLF4 expression levels in different cell lines upon IgG cross-linking as indicated.
(B) Viral DNA replication was measured by qPCR using primers to BALF5. Representative results from three biological replicates are presented. The value of a non-targeting control at 0 hr (lane 7) was set as 1. Error bars indicate SD. ∗p < 0.01.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

5. Figure 3
PIAS1 Is Downregulated following Caspase Activation upon BCR Activation
(A) Immunoblot showing PIAS1 degradation, the generation of cleaved-PARP and other cleaved caspase substrates, and the accumulation of EBV ZTA and BGLF4. Akata (EBV+) and Akata-4E3 (EBV−) cells were treated with anti-IgG antibody for 0 to 48 hr as indicated.
(B) Western blot analyses showing caspase activation upon IgG cross-linking.
(C) Caspase inhibition blocks PIAS1 degradation. The cells were either untreated or pretreated with a pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hr. Western blot was performed using antibodies as indicated.
(D) Caspase inhibition blocks PIAS1 cleavage. Akata (EBV+) cells were either untreated or pretreated with a caspase-3/7 inhibitor (Z-DEVD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hr. Western blot was performed using antibodies as indicated. Arrow denotes cleaved PIAS1.
See also Figures S2, S3, and S4.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

6. Figure 4 Caspase-3, -6, and -8 Cleave PIAS1 In Vitro
(A) Schematic representation of the Halo-V5-PIAS1. Red Ser550 denotes the epitope position for anti-PIAS1 antibody.
(B and C) V5-tagged wild-type PIAS1 was incubated with individual recombinant caspase for 2 hr at 37°C. Western blot was performed using either anti-V5 (B) or anti-PIAS1 (C) antibodies. The relative positions of predicted cleaved bands were labeled as indicated.
See also Figure S2.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

7. Figure 5 Caspase-3, -6, and -8 Cleave PIAS1 after D100 and D433
(A) Schematic representation of functional domains and two of the predicted cleavage sites of PIAS1. SAP (SAF-A/B, Acinus, and PIAS): DNA and protein binding domain; PINIT: nuclear localization motif; RING finger: E3 ligase domain for protein SUMOylation; SIM: SUMO interacting motif; S/T Rich: variable Ser/Thr rich region.
(B to G) V5-tagged D100A, D148A, D433A or D100/433A PIAS1 mutants were incubated with individual recombinant caspases for 2 hr at 37°C. WB was performed using either anti-V5 (B, D and F) or anti-PIAS1 (C, E and G) antibodies. The relative positions of cleaved bands were labeled as indicated. In (B) and (C), marker lanes were grouped from the same film to show the relative positions of cleaved fragments.
See also Figure S2 and Table S2.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

8. Figure 6
Caspase Activation Facilitates EBV Replication through PIAS1 Cleavage
(A) Caspase inhibitors and the concentration used in the following experiments.
(B–D) Caspase inhibition blocks EBV lytic replication. Akata (EBV+) cells were either untreated or pretreated with caspase inhibitors, individually or in combination for 1 hr, and then anti-IgG antibody was added for 48 hr as indicated. EBV ZTA and RTA expression and the generation of cleaved PARP were monitored by western blot (B). EBV gene expression was measured by qRT-PCR using primers as indicated (C). Relative EBV DNA copy numbers were measured by qPCR using primers specific for BALF5 and BHLF1 (D). The value of cells without treatment (lane 1) was set as 1.
(E) Caspase activation facilitates lytic replication through PIAS1 cleavage. PIAS1-depleted (sg-1) and control Akata (EBV+) cells were either untreated or pretreated with caspase inhibitors for 1 hr, and then anti-human IgG antibody was added for 48 hr. Western blot was performed using antibodies as indicated. Relative EBV DNA copy numbers were measured by qPCR using primers specific for BALF5. The value of control without treatment (lane 5) was set as 1.
(F and G) Cleavage-resistant PIAS1 further suppresses EBV replication. Akata (EBV+) sg-1 cells were used to establish PIAS1-knockout (PIAS1-KO) single-cell clones. PIAS1-KO cells were then reconstituted with wild-type (WT) or cleavage-resistant (D100/433A) PIAS1 using lentiviral constructs. Western blot analyses indicate PIAS1, ZTA, RTA, and BGLF4 expression levels in these cell lines upon IgG cross-linking as indicated (F). Viral DNA replication was measured by qPCR using primers specific to BALF5 and BHLF1 (G). The value of WT PIAS1 at 0 hr (lane 4) was set as 1.
Representative results from three biological replicates are presented. Error bars indicate SD. ∗p < 0.01.
See also Figures S2, S3, and S4.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

9. Figure 7
PIAS1 Blocks C/EBPβ-, ZTA-, and RTA-Mediated Lytic Gene Activation
(A) Suppression of Zp- and Rp-Luc reporters by PIAS1. 293T cells were transfected with 250 ng plasmid DNA encoding Zp- or Rp-Luc, and effector plasmid DNA expressing ZTA and increasing amounts of PIAS1 as indicated. The total amount of effector plasmid DNA used in each transfection was normalized by adding vector DNA.
(B) PIAS1 suppresses ZTA-C/EBPβ-mediated Zp promoter activation. 293T cells were transfected with 250 ng Zp-Luc and other plasmid DNA as indicated.
(C) PIAS1 suppresses RTA-mediated BGLF2 promoter activation. 293T cells were transfected with 250 ng BGLF2p-Luc and other plasmid DNA as indicated.
(D) PIAS1 binds to EBV promoters. The relative positions of EBV ZTA, RTA, and BHLF1 are depicted in the top panel. ChIP-PCR analysis was performed on Akata (EBV+) cells showing PIAS1 binding to ZTA (Zp) and RTA (Rp) promoters but not BHLF1 (BHLF1p) promoter in the PIAS1-expressing control cells. ChIP by a nonspecific IgG was included as a negative control.
Representative results from three biological replicates are presented. Error bars indicate the SD. ∗p < 0.01.
See also Figures S5, S6, and S7.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions