Anne Nohl Division of Hematology and Center for the Study of Blood Diseases, University of Southern California Keck School of Medicine, Los Angeles, California, USA.
J Virol. 2014 Jun;88(11):6345-54. doi: 10.1128/JVI.00028-14. Epub 2014 Mar 26.
Kaposi's sarcoma-associated herpesvirus-encoded viral FLICE inhibitory protein (vFLIP) K13 was originally believed to protect virally infected cells against death receptor-induced apoptosis by interfering with caspase 8/FLICE activation. Subsequent studies revealed that K13 also activates the NF-κB pathway by binding to the NEMO/inhibitor of NF-κB (IκB) kinase gamma (IKKγ) subunit of an IKK complex and uses this pathway to modulate the expression of genes involved in cellular survival, proliferation, and the inflammatory response. However, it is not clear if K13 can also induce gene expression independently of NEMO/IKKγ. The minimum region of NEMO that is sufficient for supporting K13-induced NF-κB has not been delineated. Furthermore, the contribution of NEMO and NF-κB to the protective effect of K13 against death receptor-induced apoptosis remains to be determined. In this study, we used microarray analysis on K13-expressing wild-type and NEMO-deficient cells to demonstrate that NEMO is required for modulation of K13-induced genes. Reconstitution of NEMO-null cells revealed that the N-terminal 251 amino acid residues of NEMO are sufficient for supporting K13-induced NF-κB but fail to support tumor necrosis factor alpha (TNF-α)-induced NF-κB. K13 failed to protect NEMO-null cells against TNF-α-induced cell death but protected those reconstituted with the NEMO mutant truncated to include only the N-terminal 251 amino acid residues [the NEMO(1-251) mutant]. Taken collectively, our results demonstrate that NEMO is required for modulation of K13-induced genes and the N-terminal 251 amino acids of NEMO are sufficient for supporting K13-induced NF-κB. Finally, the ability of K13 to protect against TNF-α-induced cell death is critically dependent on its ability to interact with NEMO and activate NF-κB.
Kaposi's sarcoma-associated herpesvirus-encoded vFLIP K13 is believed to protect virally infected cells against death receptor-induced apoptosis and to activate the NF-κB pathway by binding to adaptor protein NEMO/IKKγ. However, whether K13 can also induce gene expression independently of NEMO and the minimum region of NEMO that is sufficient for supporting K13-induced NF-κB remain to be delineated. Furthermore, the contribution of NEMO and NF-κB to the protective effect of K13 against death receptor-induced apoptosis is not clear. We demonstrate that NEMO is required for modulation of K13-induced genes and its N-terminal 251 amino acids are sufficient for supporting K13-induced NF-κB. The ability of K13 to protect against TNF-α-induced cell death is critically dependent on its ability to interact with NEMO and activate NF-κB. Our results suggest that K13-based gene therapy approaches may have utility for the treatment of patients with NEMO mutations and immunodeficiency.
卡波西肉瘤相关疱疹病毒编码的病毒 FLICE 抑制蛋白（vFLIP）K13 最初被认为通过与半胱天冬酶 8/FLICE 激活物（FLICE）相互作用来保护病毒感染的细胞免于死亡受体诱导的细胞凋亡。随后的研究表明，K13 还通过与 IKK 复合物的 NEMO/抑制 NF-κB（IκB）激酶γ（IKKγ）亚基结合来激活 NF-κB 途径，并利用该途径调节参与细胞存活、增殖和炎症反应的基因的表达。然而，目前尚不清楚 K13 是否可以独立于 NEMO/IKKγ 诱导基因表达。支持 K13 诱导的 NF-κB 的 NEMO 的最小区域尚未确定。此外，NEMO 和 NF-κB 对 K13 对死亡受体诱导的细胞凋亡的保护作用的贡献仍有待确定。在这项研究中，我们使用 K13 表达的野生型和 NEMO 缺陷细胞的微阵列分析来证明 NEMO 是调节 K13 诱导基因所必需的。NEMO 缺陷细胞的重建表明，NEMO 的 N 端 251 个氨基酸残基足以支持 K13 诱导的 NF-κB，但不能支持肿瘤坏死因子α（TNF-α）诱导的 NF-κB。K13 不能保护 NEMO 缺陷细胞免受 TNF-α诱导的细胞死亡，但可保护那些用仅包含 N 端 251 个氨基酸残基的 NEMO 突变体（NEMO（1-251）突变体）重建的细胞。总的来说，我们的结果表明，NEMO 是调节 K13 诱导基因所必需的，并且 NEMO 的 N 端 251 个氨基酸残基足以支持 K13 诱导的 NF-κB。最后，K13 保护细胞免受 TNF-α诱导的细胞死亡的能力严重依赖于其与 NEMO 相互作用和激活 NF-κB 的能力。
卡波西肉瘤相关疱疹病毒编码的 vFLIP K13 被认为可以保护病毒感染的细胞免于死亡受体诱导的细胞凋亡，并通过与衔接蛋白 NEMO/IKKγ 结合来激活 NF-κB 途径。然而，K13 是否也可以独立于 NEMO 并在 NEMO 中确定足以支持 K13 诱导的 NF-κB 的最小区域仍有待确定。此外，NEMO 和 NF-κB 对 K13 对死亡受体诱导的细胞凋亡的保护作用的贡献尚不清楚。我们证明 NEMO 是调节 K13 诱导基因所必需的，其 N 端 251 个氨基酸残基足以支持 K13 诱导的 NF-κB。K13 保护细胞免受 TNF-α诱导的细胞死亡的能力严重依赖于其与 NEMO 相互作用和激活 NF-κB 的能力。我们的结果表明，基于 K13 的基因治疗方法可能对治疗 NEMO 突变和免疫缺陷的患者有用。
