Kaposi’s Sarcoma Associated Herpesvirus Encoded Viral FLICE Inhibitory Protein K13 Activates NF-κB Pathway Independent of TRAF6, TAK1 and LUBAC

BACKGROUND: Kaposi's sarcoma associated herpesvirus encoded viral FLICE inhibitory protein (vFLIP) K13 activates the NF-κB pathway by binding to the NEMO/IKKγ subunit of the IκB kinase (IKK) complex. However, it has remained enigmatic how K13-NEMO interaction results in the activation of the IKK complex. Recent studies have implicated TRAF6, TAK1 and linear ubiquitin chains assembled by a linear ubiquitin chain assembly complex (LUBAC) consisting of HOIL-1, HOIP and SHARPIN in IKK activation by proinflammatory cytokines. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that K13-induced NF-κB DNA binding and transcriptional activities are not impaired in cells derived from mice with targeted disruption of TRAF6, TAK1 and HOIL-1 genes and in cells derived from mice with chronic proliferative dermatitis (cpdm), which have mutation in the Sharpin gene (Sharpin(cpdm/cpdm)). Furthermore, reconstitution of NEMO-deficient murine embryonic fibroblast cells with NEMO mutants that are incapable of binding to linear ubiquitin chains supported K13-induced NF-κB activity. K13-induced NF-κB activity was not blocked by CYLD, a deubiquitylating enzyme that can cleave linear and Lys63-linked ubiquitin chains. On the other hand, NEMO was required for interaction of K13 with IKK1/IKKα and IKK2/IKKβ, which resulted in their activation by "T Loop" phosphorylation. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that K13 activates the NF-κB pathway by binding to NEMO which results in the recruitment of IKK1/IKKα and IKK2/IKKβ and their subsequent activation by phosphorylation. Thus, K13 activates NF-κB via a mechanism distinct from that utilized by inflammatory cytokines. These results have important implications for the development of therapeutic agents targeting K13-induced NF-κB for the treatment of KSHV-associated malignancies

HOIL-1 is not essential for K13-induced NF-κB activation.

<p><b>A.</b> The expression of FLAG-tagged K13-ER^TAM in wild-type and <i>HOIL-1^−/−</i> MEF was confirmed with Western blotting. The blot was re-probed with a tubulin antibody to show equal protein loading. <b>B.</b> Wild-type and <i>HOIL-1^−/−</i> MEFs stably expressing FLAG-K13-ER^TAM were transfected with NF-κB-Luc and Renilla reporter constructs. Cells were subsequently treated with 4OHT (20 nM) for 48 hours and the luciferase reporter assay was performed essentially as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036601#pone-0036601-g001" target="_blank">Figure 1A</a>. Asterisks (*) indicate significance at levels of p≤0.05 as compared to vehicle-treated cells. <b>C.</b> Wild-type and <i>HOIL-1^−/−</i> MEFs were transfected with NF-κB-Luc and Renilla reporter constructs and 6 hours post-transfection, these cells were treated with mTNF-α (10ng/ml) for 18 hours and the luciferase reporter assay was performed essentially as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036601#pone-0036601-g001" target="_blank">Figure 1A</a>. <b>D.</b> Expression of transduced FLAG-tagged K13 in wild-type and <i>HOIL-1^−/−</i> MEFs was examined by immunoblot analysis; tubulin was used as a loading control. <b>E.</b> Nuclear p65 DNA binding activities in the nuclear extracts of wild-type and <i>HOIL-1^−/−</i> MEFs expressing an empty vector or FLAG-K13. Asterisks (*) indicate significance at levels of p≤0.05 as compared to vector cells. <b>F.</b> Nuclear p65 DNA binding activities in the nuclear extracts of wild-type and <i>HOIL-1^−/−</i> MEFs following treatment with murine TNFα. <b>G.</b> Wild-type and <i>HOIL-1^−/−</i> MEFs expressing FLAG-K13 were examined for NF-κB activation by Western blot analysis using antibodies against phospho-IκBα, Total IκBα, A20 and RelB. The blot was re-probed with FLAG and Tubulin antibodies to check the expression of the transduced K13 and equal protein loading, respectively.</p

SHARPIN is not required for K13-induced NF-κB activation.

<p><b>A.</b> MEFs deficient in SHARPIN (cpdm) were able to activate NF-κB reporter activity in the presence of K13, but not with TNFα. Asterisks (*) indicate significance at levels of p≤0.05. <b>B.</b> SHARPIN is not required for K13-induced NF-κB p65 DNA binding as determined by ELISA. Asterisks (*) indicate significance at levels of p≤0.05 as compared to vehicle-treated cells. <b>C.</b> Total cell lysates from wild-type, and <i>cpdm</i> MEF stably expressing an empty vector or FLAG-tagged K13 were used to examine the requirement of SHARPIN on K13-induced NF-κB activation by upregulation of expression of A20, RelB, phosphorylation of IκBα and degradation of total IκBα. The expression of the FLAG-tagged K13 protein and equal protein loading was confirmed by blotting with FLAG and Tubulin antibodies, respectively.</p

Deubiquitinating enzyme CYLD, a negative regulator of NF-κB pathway does not block K13-induced NF-κB activity. A–D.

<p>The 293T cells were co-transfected with a control vector or vectors encoding FLAG-K13, TNFR1, CD40 and EDAR (250 ng/ml) and a CYLD plasmid construct (750 ng/well) along with NF-κB-Luc reporter construct (75 ng/well) and a pRSV/LacZ (β-galactosidase) reporter construct (75 ng/well), and the reporter assay performed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036601#s2" target="_blank">Materials and Methods</a>” The values shown are averages (mean±SE) of one representative experiment out of three in which each transfection was performed in duplicate.</p

TRAF6 is not required for K13-induced NF-κB activation.

<p><b>A... </b><i>TRAF6^+/+</i> and <i>TRAF6^−/−</i> MEFs were transfected with a control vector or vector encoding K13 along with an NF-κB-Luc construct (75 ng/well) and a Renilla reporter construct (75 ng/well, normalization control) using Lipofectamine 2000-mediated transaction. The luciferase reporter assay was performed 48 hours post transfection essentially as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036601#s2" target="_blank">Materials and Methods</a>” section. Asterisks (*) indicate significance at levels of p≤0.05. <b>B... </b><i>TRAF6^+/+</i> and <i>TRAF6^−/−</i> MEFs were transfected with a control vector or a vector encoding K13-ER^TAM along with an NF-κB-Luc construct and a Renilla reporter construct. The transfected cells were subsequently treated with 4OHT (20 nM) for 48 hours and the reporter assays performed as described above. The values shown are averages (mean±SE) of one representative experiment out of three in which each transfection was performed in duplicate. Asterisks (*) indicate significance at levels of p≤0.05. <b>C... </b><i>TRAF6^+/+</i> and <i>TRAF6^−/−</i> MEF cells expressing an empty vector or the FLAG-tagged K13-ER^TAM treated with 4OHT were examined by immunoblot analysis for upregulation of A20. Tubulin was used as a loading control.</p

K13 activates NF-κB pathway without involving TAK1. A.

<p>Immunoblot showing expression of ectopically expressed FLAG-tagged K13 in <i>TAK1^+/+</i> and <i>TAK1^−/−</i> MEFs and endogenous K13 in BC1 cell line. <b>B.</b> Status of NF-κB pathway, as measured by an EMSA in <i>TAK1^+/+</i> and <i>TAK1^−/−</i> MEF cells stably expressing vector and K13. The position of the induced NF-κB complexes is marked by an <i>asterisk</i>, while an <i>arrow</i> marks the position of the constitutive complexes. <b>C.</b> The NF-κB subunits composition of the K13-induced nuclear NF-κB complexes in the wild-type and <i>TAK1^−/−</i> MEFs was determined with an ELISA-based DNA-binding assay performed in triplicate. Asterisks (*) indicate significance at levels of p≤0.05. <b>D. </b><i>TAK1^+/+</i> and <i>TAK1^−/−</i> MEFs expressing FLAG-K13-ER^TAM were transfected with NF-κB-Luc and Renilla reporter constructs and subsequently treated with 4OHT (20 nM) for 48 hours and the reporter assays performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036601#pone-0036601-g001" target="_blank">Figure 1A</a>. Asterisks (*) indicate significance at levels of p≤0.05. <b>E.</b> Nuclear extracts from Jurkat, BC1, BCBL1 and Namalwa were used for the measurement of p65 DNA-binding activity using an ELISA-based assay. <b>F.</b> Lack of phosphorylation of TAK1 by K13. Cell lysates prepared from BCBL1 and Namalwa cells expressing an empty vector or FLAG-K13 were probed with an antibody to detect phosphorylation of TAK1. Vector cells treated with TNFα were used a positive control. The blot was re-probed with FLAG and Tubulin antibodies to check the expression of the transduced K13 and equal protein loading, respectively. <b>G</b>−<b>H.</b> Treatment with TAK1 inhibitor 5Z-7-oxo-zeaenol had no effect on 4OHT induced NF-κB-Luc activity in 293-K13-ER^TAM-NF-κB-Luc cells but effectively blocked TNFα and IL1β induced NF-κB-Luc activity.</p