Figure 1.
Purification and identification of LANA binding proteins.
(A) Outline of biochemical purification baits all of which pulled down Hsp90; (1.) wild-type, unmodified LANA; (2.) as used to derive partners listed in table 1; (3.) as used to derive partners listed in table 2. The abbreviations denote epitopes: F for DYKDDDDK, HA for YPYDVPDYA and EQEQE which is repeated within the LANA central domain. (B) Schematic procedure for purification of LANA complexes using bait 2. Samples were eluted from columns of Sepharose 6B and Heparin FF for purification separately, followed by immunoaffinity purification with mouse anti-Flag M2 affinity gel and immunoprecipitated using anti-LANA mab. (C) SDS-PAGE analysis was performed by 8–16% gradient gel and stained with colloidal blue. (D) Schematic procedure for purification of LANA mutant complexes using bait 3. (E) SDS-PAGE analysis was performed by 8–16% gradient gel and stained with colloidal blue. Co-IP, immunoprecipitation; M, molecular mass (in kDa).
Table 1.
Result of MS/MS for Flag-LANA associated proteins (<1,000 aa).
Table 2.
Result of MS/MS for HA-Flag-LANA-dCR (pMF-24) associated proteins (<1,000 aa).
Figure 2.
Analysis of interaction between LANA and Hsp90.
(A) Interaction between Hsp90 and LANA or respective LANA mutants in HeLa cells. HA-tagged Hsp90 was co-transfected with Flag-tagged full-length LANA (aa1–1162) or indicated mutants: Flag-tagged LANA-N (aa1–329, pDD1928), Flag-tagged LANA-C(aa930–1162,pDD1931), or Flag-tagged LANA N+C (aa1–329 and 928–1162, pDD775). Protein extracts were immunoprecipitated with anti-Flag antibody followed by immunoblotting with anti-HA antibodies, IgG was used as control. Input samples were from cell lysate supernatants. MW markers in kD are indicated on the left. (B) Reverse immunoprecipitation. HA-tagged Hsp90 together with Flag-tagged LANA were co-transfected into HeLa cells, empty pcDNA vector was used for control. Protein extracts were immunoprecipitated with anti-HA antibody and immunoblotted with anti-LANA antibody. Input samples were from cell lysate supernatant. (C) Co-localization analysis of LANA and Hsp90. Immunofluorescence assay was performed after fixation and permeabilization of TIVE-L1 cells, incubated with primary rabbit anti-LANA and mouse anti-Hsp90 antibodies and the secondary anti-rabbit Texas (red) and anti-mouse FITC (green) conjugated antibodies respectively. Nuclear fractions were stained with DAPI, images were observed under fluorescence microscope.
Figure 3.
Hsp90 inhibitors disassociate LANA and Hsp90.
(A) BCBL-1 cells were harvested after treatment with 0.5 µM 17-DMAG for 0, 3, 6, 12, 24 hours separately, cells lysates were immunoprecipitated with rat monoclonal anti-LANA antibody, followed by immunoblotting analysis with mouse anti-Hsp90 and anti-LANA antibodies. Input samples were from supernatants of lysed cells. (B) BCBL-1 cells were harvested after treatment with 0, 10, 100 and 1000 µM AUY922 separately for 24 hours, cell lyses were used for immunoprecipitation assay with anti-Hsp90 and anti-p53 antibodies, followed by immunoblotting analysis with anti-LANA. (C) Hela cells were transfected with LANA vector treated with no drug (DMSO) or 0.1 µM AUY922 for 24 hours. Immunoprecipitation was performed with rat anti-LANA antibody, followed by immunoblotting analysis with anti-LANA and anti-Hsp90 antibodies; IgG was used as control.
Figure 4.
Hsp90 inhibitors induce proteasomal degradation of LANA.
(A) Half-life analysis of LANA termini. N- or C- termini of Flag-tagged LANA vectors were transfected separately into HeLa cells for 16 hours, followed by cycloheximide (CHX) treatment for 0, 3, 6, 12, 24 and 48 hours. Whole cells lysates were immunoblotted with anti-Flag antibody. β-Actin was used for loading control. (B) LANA degradation induced by 17-DMAG. HeLa cells were transfected with full-length LANA overnight, followed by incubation with vehicle or 0.5 µM 17-DMAG in the presence of 50 µg/ml cycloheximide for 0, 3, 6, 12, 24 and 48 hours respectively, whole cells lysates were immunoblotted with anti-LANA. (C–D) Quantitative analysis of the above results. (E–F) LANA degradation inhibited by MG-132 or Lactacystin. After transfection with LANA vector, Hela cells were treated with no drug or 17-DMAG (1 µM) for 24-hours in the absence (−) or presence (+) of proteasome inhibitor MG-132 (10 µM) for the last 6 hours or Lactacystin (10 µM) for 24 hours, whole cells lyses were immunoblotted with anti-LANA antibody. (G) Proteasomal degradation of LANA in BCBL-1 cells. BCBL-1 were treated for 24 hours with no drug or AUY922 (0.1 µM) in the absence (−) or presence (+) of proteasome inhibitor MG-132 (10 µM) for the last 6 hours, whole cells lysates were immunoblotted with anti-LANA antibody. (H) Poly-ubiquitinated degradation of LANA. HeLa cells were transfected with LANA, followed by treatment with no drug or 1 µM 17-DMAG for 24 hours in the presence of DMSO or 10 µM MG-132 for the last 6 hours. Cells lysates were immunoprecitated with rat anti-LANA antibody, followed by immunoblotting with rabbit anti-ubiquitin and anti-LANA antibodies, the bracket shows the poly-ubiquitinated LANA (Ub-LANA). (I) Immunofluorescence analysis of LANA degradation. L1T2 cells were treated with no drug or 1 µM 17-DMAG for 48 hours, incubated with primary anti-LANA (rabbit) and after fixation and permeabilization, stained with anti-rabbit Texas-Red conjugated antibodies (red, LANA), nuclei were stained with DAPI.
Figure 5.
Central repeat domain of LANA resists degradation.
(A–C) HeLa cells were transfected with plasmids including full-length LANA, LANA mutant (deleted repeat central domain) and EBNA1 respectively in six-well plates, followed by treatment with 17-DMAG at concentrations of 0, 0.1, 0.5, 2.5 and 10 µM for 48 hours. Whole cells lysates of each sample were immunoblotted with anti-LANA and anti-Flag antibodies, Cdc2 and β-Actin were used as controls.
Figure 6.
Effects of Hsp90 inhibitors on LANA in PEL cells.
(A–D) Hsp90 inhibitors repress LANA expression in PEL cells. PEL cells including BC-3, BCP-1, BCBL-1, and BC-1 cells were treated with 17-DMAG at concentrations of 0, 0.1, 0.5, 2.5 and 10 µM and after 48 hours, whole cells lysates were immunoblotted with anti-LANA, anti-Hsp90, anti-Akt, and anti-cleaved caspase-3 antibodies, Cdc2 and β-Actin were used as controls. (E–G) Apoptosis. PEL cells including BC-3, BCP-1, BCBL-1, and BC-1 cells were treated respectively with Hsp90 inhibitors 17-DMAG and PU-H71 at concentrations of 0, 1, and 10 µM, and 10 µM, or 0, 0.1 µM AUY922 for 24 hours, apoptosis percentage was analyzed after PEL cells were harvested and stained.
Figure 7.
Effects of Hsp90 on LANA after lentiviral knockout.
BCBL-1 and BC-1 cells were infected with recombinant lentiviruses targeting Hsp90 in six-well plates; empty lentivirus vector and untreated cells were used as controls. Samples were harvested after 4 days and immunoblotted with anti-LANA, anti-Hsp90, and anti-Akt antibodies, respectively. Apoptosis was evaluated by immunoblotting assay with anti-cleaved PARP and Caspase-3 antibodies separately. β-Actin was used as loading control.
Figure 8.
Effects of Hsp90 inhibitors on LANA in KSHV-infected endothelial cells.
(A) L1T2 cells were treated with AUY922 at concentrations of 0, 0.02, 0.1, 0.5, and 2.5 µM for 48 hours, whole cell lysates were immunoblotted with anti-LANA, anti-Hsp90 antibodies, anti-EphA2, anti-Ephrin-B2, and anti-Akt (total), and anti-pAkt (S473) antibodies separately. Apoptosis was evaluated with anti-cleaved PARP and anti-cleaved Caspase-3 antibodies, Cdc2 and β-Actin were used as controls. (B–C) SLK-KSHV were treated with 0.5 µM 17-DMAG for 0, 12, 24, 48, 72 and 96 hours, or at concentrations of 0, 0.1, 0.5, 2.5 and 10 µM for 48 hours separately, whole cell lysates were immunoblotted with anti-LANA antibody, anti-EphA2, anti-EphrinB2, Cdc2 and β-Actin were used as controls.
Figure 9.
Hsp90 inhibitors repress KS tumor growth.
(A) Colony formation assay of L1T2 cells after drug treatment. 400 L1T2 cells were seeded in 10 cm dishes, followed by addition of two-fold serially-diluted Hsp90 inhibitors; 17-DMAG, PU-H71, AUY922, BIIB021, and NVP-BEP800, respectively for two weeks. Colonies were counted after incubation with Magic Blue Staining, each experiment was repeated three times. (B) Hsp90 inhibitors induce G0/G1 arrest of L1T2 cells. L1T2 cells were treated with 0.5 µM of 17-DMAG, PU-H71, NVP-BEP800, BIB021, or 0.05 µM AUY922 for 24 hours, DMSO treatment was used as control. After cells were fixed and stained with propidium iodide, cell-cycle analysis was performed using flow cytometry. The percentages of cells at different stages in the cell cycle (G0/G1, S, G2/M) are shown. (C) Growth curves of tumor volume. 105 L1T2 cells were injected sub-cutaneously into C.B.-17 SCID mice with mixed Matrigel (1∶1) for three days, followed by AUY922 intra-peritoneal injection at doses of 50 mg/kg NVP-AUY922 for total three weeks (three times per week), tumor volumes of SCID mice were measured and analyzed. Two groups were analyzed and each group had six mice, mock-treated mice were used as control.
Table 3.
IC50 based on Xcelligence measurements of cell proliferation.
Figure 10.
Immunohistochemistry analysis of mouse Xenograft tumors.
Solid tumors were excised from mock or drug treated mice, followed by immunohistochemistry staining using antibodies specific for p-Akt, LANA, and Ephrin B2. No specific primary antibody was used for control. Images were taken at 100× and 400× magnification.