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. 2013 Feb;87(4):2242-52.
doi: 10.1128/JVI.01393-12. Epub 2012 Dec 12.

Kaposi's sarcoma-associated herpesvirus oncoprotein K13 protects against B cell receptor-induced growth arrest and apoptosis through NF-κB activation

Ciaren Graham  1 , Hittu Matta, Yanqiang Yang, Han Yi, Yulan Suo, Bhairavi Tolani, Preet M Chaudhary
Affiliations

Kaposi's sarcoma-associated herpesvirus oncoprotein K13 protects against B cell receptor-induced growth arrest and apoptosis through NF-κB activation

Ciaren Graham et al. J Virol. 2013 Feb.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) has been linked to the development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease (MCD). We have characterized the role of KSHV-encoded viral FLICE inhibitory protein (vFLIP) K13 in the modulation of anti-IgM-induced growth arrest and apoptosis in B cells. We demonstrate that K13 protects WEHI 231, an immature B-cell line, against anti-IgM-induced growth arrest and apoptosis. The protective effect of K13 was associated with the activation of the NF-κB pathway and was deficient in a mutant K13 with three alanine substitutions at positions 58 to 60 (K13-58AAA) and a structural homolog, vFLIP E8, both of which lack NF-κB activity. K13 upregulated the expression of NF-κB subunit RelB and blocked the anti-IgM-induced decline in c-Myc and rise in p27(Kip1) that have been associated with growth arrest and apoptosis. K13 also upregulated the expression of Mcl-1, an antiapoptotic member of the Bcl2 family. Finally, K13 protected the mature B-cell line Ramos against anti-IgM-induced apoptosis through NF-κB activation. Inhibition of anti-IgM-induced apoptosis by K13 may contribute to the development of KSHV-associated lymphoproliferative disorders.

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Figures

Fig 1
Fig 1
K13 protects WEHI 231 cells from anti-IgM induced growth inhibition. (A) Expression of FLAG-K13 in WEHI 231 cells. K13 was immunoprecipitated (IP) by FLAG antibody beads from WEHI 231 cells stably expressing an empty vector or FLAG-tagged K13 followed by immunoblot (IB) analysis with a FLAG antibody. (B) Level of ectopic K13 in WEHI 231 cells compared with its physiological level in the KSHV-infected PEL cell line BC1. Immunoblotting was performed using 8F6 monoclonal antibody against K13; a nonspecific band (NS) serves as a loading control. (C) WEHI 231 cells expressing an empty vector or K13 were grown in triplicate in a 96-well plate with the indicated concentrations of anti-IgM for 48 h. Cell viability was measured using the MTS assay (values are means ± SD; n = 3). (D) Expression of FLAG-K13-ERTAM in WEHI 231 cells, as measured by Western blotting. (E) WEHI 231 cells expressing K13-ERTAM grown in the presence and absence of 4OHT (20 nM) were treated in triplicate with the indicated concentrations of anti-IgM for 48 h. Cell viability was measured using the MTS assay (values are means ± SD; n = 3).
Fig 2
Fig 2
K13 protects WEHI 231 cells from anti-IgM-induced growth arrest and apoptosis. (A) WEHI 231-K13-ERTAM cells were grown in the presence and absence of 4OHT (20 nM) and then treated with and without anti-IgM (1 μg/ml) for 48 h. Cells were stained with propidium iodide, and cell cycle analysis was performed using flow cytometry. (B) WEHI 231-K13-ERTAM cells grown in the presence or absence of 4OHT (20 nM) were stained with phycoerythrin-labeled annexin V after treatment with anti-IgM (1 μg/ml) for 48 h and examined by flow cytometry. (C) WEHI 231-K13-ERTAM cells grown in the presence and absence of 4OHT were treated with 1 μg/ml of anti-IgM. Cells were then stained with Sytox Green, a cell-impermeable nuclear dye that stains the nuclei of dead cells; cells were then examined under a fluorescence microscope or under phase-contrast microscope and photographed.
Fig 3
Fig 3
K13 activates the NF-κB pathway in WEHI 231 cells. (A) An ELISA-based NF-κB binding assay showing increased binding of p65/RelA DNA binding activity in the nuclear extracts of WEHI 231-vector and WEHI 231-K13-ERTAM cells grown in the presence or absence of 4OHT and treated with 1 μg/ml of anti-IgM for 24 h. Values shown are the mean ± SD from one representative experiment out of three performed in duplicate. Asterisks indicate significance at a P level of ≤0.05. OD 655nm, optical density at 655 nm. (B) A luciferase-based reporter assay showing induction of NF-κB transcriptional activity by K13. WEHI 231 cells stably expressing the K13-ERTAM-NF-κB-Luc construct were grown in the presence and absence of 4OHT and treated with 1 μg/ml of anti-IgM for 24 h, and cell lysates were used for a luciferase reporter assay. Values shown are the mean ± SD from one representative experiment out of three performed in duplicate. Asterisks indicate significance at a P level of ≤0.05. (C) Western blots showing upregulation of A20 and RelB expression in 4OHT-treated WEHI 231 K13-ERTAM cells and no significant decline following treatment with anti-IgM (1 μg/ml). WEHI 231-vector cells treated with 4OHT did not show any increase in A20 and RelB. Tubulin served as a loading control. (D) Wild-type K13, but not vFLIP E8 or K13-58AAA, induces RelB promoter activity. 293T cells were transfected with a control vector and a vector encoding wild-type K13 or the K13 mutant K13-58AAA or vFLIP E8 (250 ng/ml) along with an RelB-Luc reporter construct (75 ng/well) and a pRSV/LacZ (β-galactosidase) reporter construct (75 ng/well), and the reporter assay was performed as described in Materials and Methods. The values shown are mean ± SD of one representative experiment out of three in which each transfection was performed in duplicate. (E) Expression levels of wild-type FLAG-tagged K13, the K13 mutant K13-58AAA, and vFLIP E8 in 293T cells. (F) K13 activates RelB promoter through NF-κB the pathway. 293T cells were transfected with a control vector or a vector encoding K13 along with either a WT-RelB-Luc or RelB-Luc construct containing mutations in NF-κB RE I (Mut I), NF-κB RE II (Mut II), or both (Mut 1+II). The experiment was performed as described in for panel D. (G) Dominant negative mutants of IκBα lacking the N-terminal 36 amino acids (IκBαΔN) and a superrepressor form of IκBα (IκBα SS32/36AA) block K13-induced RelB promoter activity. 293T cells were transfected either with an empty vector or K13, along with an RelB luciferase reporter construct and a pRSV/LacZ reporter construct, as described for panel F. The amount of inhibitor plasmids (500 ng/well) was 5 times the amount of vector or K13 (100 ng/well) plasmid, and the total amount of transfected DNA was kept constant by adding an empty vector. The values shown are the mean ± SD of one representative experiment out of three in which each transfection was performed in duplicate. (H) As2O3, an inhibitor of NF-κB, blocks K13-induced RelB promoter activation. 293T cells were transfected with an empty vector or a vector encoding K13 along with RelB-Luc and pRSV/LacZ reporter constructs. Approximately 3 h after transfection, cells were treated with control vehicle or 2.5 μM As2O3 for 18 h before cell lysis and measurement of reporter activities.
Fig 4
Fig 4
The protective effect of K13 against anti-IgM-induced apoptosis is dependent on NF-κB signaling. (A) Expression of K13, K13-58AAA, and vFLIP E8 in WEHI 231 cells as determined by immunoprecipitation with FLAG beads followed by immunoblot analysis with a FLAG antibody. The asterisk denotes a nonspecific band. (B) Western blot analysis showing expression of A20 and RelB in WEHI 231 cells expressing wild-type K13, K13-58AAA, and vFLIP E8. GAPDH served as a loading control. The blot was imaged using an Odyssey Infrared Imaging System. (C) WEHI 231 cells expressing wild-type K13, K13-58AAA, and the vFLIP E8 were subjected to anti-IgM treatment at the indicated concentrations. Cell viability was measured using an MTS assay (values are means ± SD; n = 3). (D) Western blot analysis showing siRNA-mediated knockdown of RelB expression in WEHI 231-K13 cells. The blot was reprobed with an antibody against GAPDH (bottom panel) to show equal loading and specificity of gene silencing. The blot was imaged using an Odyssey Infrared Imaging System. G3PDH, glyceraldehyde-3-phosphate dehydrogenase. (E and F) Downregulation of RelB in the WEHI 231-K13 and WEHI 231-K13-ERTAM cells by siRNA results in a significant reduction in cell viability following anti-IgM (1 μg/ml) treatment compared to the control siRNA-transfected cells. The experiment was performed as described in the legend of Fig. 3C. Values shown are the means ± SD from one representative experiment out of two performed in triplicate. Asterisks indicate statistical significance at a P level of ≤0.05.
Fig 5
Fig 5
K13 exerts its protective effect against anti-IgM-induced apoptosis through modulation of c-Myc and p27Kip1 levels. (A) Real-time RT-PCR analysis showing decline in c-myc levels in WEHI 231-K13 cells following treatment with anti-IgM for 24 h that was blocked by induction of K13 activity by treatment with 4OHT. Results shown are the means ± SD (n = 2). (B) Western blot analysis showing expression of c-Myc in the WEHI 231 cells expressing wild-type K13, K13-58AAA, and vFLIP E8. G3PDH served as a loading control. Blots were imaged using an Odyssey Infrared Imaging system. (C) Immunoblot analysis of WEHI 231-K13 cells grown in the presence and absence of 4OHT (20 nM) when treated with anti-IgM (1 μg/ml) for 24 h. K13 blocks anti-IgM-induced downregulation of c-Myc. Tubulin served as a loading control. (D) Immunoblot analysis with Odyssey Infrared Imaging System showing siRNA-mediated knockdown of c-Myc expression in WEHI 231-K13 cells. (E and F) Silencing of c-Myc in the WEHI 231-K13 and WEHI 231-K13-ERTAM cells results in a significant reduction in cell viability following anti-IgM treatment compared to the control siRNA-transfected cells. Values shown are the means ± SD from one representative experiment out of two performed in triplicate. (G) Western blot analysis showing suppression of c-Myc expression by a 24-h treatment with the indicated doses of JQ1. The G3PDH blot shows equal loading. (H) WEHI 231-K13 cells treated with JQ1 show a reduction in cell viability following anti-IgM (1 μg/ml) treatment. (I) Immunoblot analysis of WEHI 231-K13 cells grown in the presence and absence of 4OHT (20 nM) when treated with anti-IgM (1 μg/ml) for 24 h. K13 blocks anti-IgM-induced upregulation of p27Kip1. Tubulin served as a loading control.
Fig 6
Fig 6
K13 exerts its protective effect against anti-IgM-induced apoptosis through upregulation of Mcl-1. (A) Immunoblot showing expression of Mcl-1 in K13-ERTAM cells that had been left untreated or treated with 4OHT and then exposed to anti-IgM (1 μg/ml) over a 48-h time period. Tubulin served as a loading control. (B) Immunoblot analysis showing ectopic expression of Mcl-1 in WEHI 231 cells. Tubulin served as a loading control. (C) WEHI 231 cells expressing an empty vector or Mcl-1 were grown in triplicate in a 96-well plate in the presence or absence of anti-IgM (1 μg/ml). Cell viability was measured 48 h later using an MTS assay (values are means ± SD; n = 3). (D) Western blot analysis showing siRNA-mediated knockdown of Mcl-1 expression in WEHI 231-K13 cells. (E and F) Downregulation of Mcl-1 in the WEHI 231-K13 and WEHI 231-K13-ERTAM cells by siRNA results in a significant reduction in cell viability following anti-IgM treatment compared to the control siRNA-transfected cells. Values shown are the means ± SD from one representative experiment out of two performed in triplicate. Asterisks indicate statistical significance at a P level of ≤0.05.
Fig 7
Fig 7
K13 protects against anti-IgM-induced apoptosis in the mature B-cell line Ramos through activation of the NF-κB pathway. (A) Expression of retrovirally expressed K13-FLAG in Ramos cells and endogenous K13 in BC1 cells as determined by immunoblotting with 8F6 monoclonal antibody. Tubulin serves as a loading control. (B) Ramos cells expressing an empty vector or K13 were grown in triplicate in a 96-well plate with anti-IgM (30 μg/ml). Cell viability was measured using the MTS assay (values are means ± SD; n = 3). Asterisks indicate significance at a P level of ≤0.05. (C) Ramos cells expressing an empty vector or K13 were treated with or without anti-IgM (30 μg/ml) for 24 h. Cells were stained with propidium iodide, and cell cycle analysis was performed using flow cytometry. Values shown are the percentage of cells in the different phases of the cell cycle. (D) An ELISA-based NF-κB binding assay showing p65/RelA DNA binding activity in the nuclear extracts of Ramos cells expressing an empty vector or K13 treated with or without 30 μg/ml of anti-IgM for 24 h. The values shown are means ± SD of one representative experiment out of three in which p65 DNA binding was measured in duplicate. Asterisks indicate significance at a P level of ≤0.05. (E) Immunoblotting showing expression of RelB, Mcl-1, and Bcl2 in Ramos cells expressing an empty vector or K13 when treated with anti-IgM (30 μg/ml) over a 48-h time period.
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