The Major Component of IκBα Proteolysis Occurs Independently of the Proteasome Pathway in Respiratory Syncytial Virus-Infected Pulmonary Epithelial Cells

Previously we showed that infection of human type II airway epithelial (A549) cells with purified respiratory syncytial virus (pRSV) induced interleukin-8 transcription by a mechanism involving cytokine-inducible cytoplasmic-nuclear translocation of the RelA transcription factor. In unstimulated cells, RelA is tethered in the cytoplasm by association with the IκB inhibitor and can be released only following IκB degradation. In this study, we examined the spectrum of IκB isoform expression and kinetics of proteolysis of the isoforms in A549 cells following pRSV infection. In contrast to the rapid and robust activation of RelA DNA binding that peaked within 15 min of treatment produced by the prototypic activator tumor necrosis factor alpha (TNF-α), pRSV produced a weaker increase in RelA binding that began at 3 h and did not peak until 24 h after infection. A549 cells expressed the IκB inhibitory subunits IκBα, IκBβ, and p105; however, following either stimulus, only the IκBα and IκBβ steady-state levels declined in parallel with the increase in RelA DNA-binding activity. The >120-min half-life of IκBα in control cells was shortened to 5 min in TNF-α-stimulated cells and to 90 min in pRSV-infected cells. Although IκBα was resynthesized within 30 min following recombinant human TNFα treatment due to a robust 25-fold increase of IκBα mRNA expression (the RelA:IκBα positive feedback loop), following pRSV infection, there was no reaccumulation of IκBα protein, as infected cells produced only a 3-fold increase in IκBα mRNA at 24 h, indicating the RelA:IκBα positive feedback loop was insufficient to restore control IκBα levels. IκBα proteolysis induced by TNF-α occurred through the 26S proteasome, as both 26S proteasome activity and IκBα proteolysis were blocked by specific inhibitors lactacystin, MG-132, and ZLLF-CHO. Although total proteasome activity in 24-h pRSV-infected lysates increased twofold, its activity was >90% inhibited by the proteasome inhibitors; surprisingly, however, IκBα proteolysis was not. We conclude that RSV infection produces IκBα proteolysis through a mechanism primarily independent of the proteasome pathway.

既往研究表明，用纯化呼吸道合胞病毒（purified respiratory syncytial virus, pRSV）感染人类II型气道上皮A549细胞，可通过涉及RelA转录因子（RelA transcription factor）的细胞因子诱导性胞质-核转位机制，诱导白细胞介素-8（interleukin-8）的转录。在未受刺激的细胞中，RelA转录因子通过与IκB抑制剂（IκB inhibitor）结合而被锚定在胞质中，仅当IκB发生降解后才能被释放。本研究中，我们针对pRSV感染后A549细胞内的IκB亚型（IκB isoform）表达谱及各亚型的蛋白水解动力学展开了探究。
与经典激活剂肿瘤坏死因子α（tumor necrosis factor alpha, TNF-α）所引发的、于处理后15分钟内达到峰值的快速且强烈的RelA DNA结合激活不同，pRSV仅能引起RelA结合活性的小幅升高：该升高始于感染后3小时，直至感染后24小时才达到峰值。A549细胞表达IκB抑制亚基IκBα、IκBβ及p105；然而，在两种刺激处理后，仅IκBα与IκBβ的稳态水平随RelA DNA结合活性的升高出现同步下降。
对照组细胞中IκBα的半衰期超过120分钟，经TNF-α刺激后该半衰期缩短至5分钟，而在pRSV感染的细胞中则缩短至90分钟。尽管经重组人TNF-α处理后，由于IκBα mRNA表达出现高达25倍的上调（即RelA:IκBα正反馈环路），IκBα可在30分钟内重新合成，但pRSV感染后并未出现IκBα蛋白的重新积累：感染细胞仅在24小时时使IκBα mRNA出现3倍的上调，这表明RelA:IκBα正反馈环路不足以恢复至对照组的IκBα水平。
TNF-α诱导的IκBα蛋白水解依赖于26S蛋白酶体（26S proteasome），因为26S蛋白酶体活性与IκBα蛋白水解均可被特异性抑制剂乳胞素（lactacystin）、MG-132及ZLLF-CHO阻断。尽管感染pRSV 24小时后的细胞裂解液中总蛋白酶体活性升高了两倍，且该活性可被蛋白酶体抑制剂抑制超过90%，但令人意外的是，IκBα的蛋白水解并未受到影响。
综上，我们得出结论：呼吸道合胞病毒感染引发的IκBα蛋白水解，主要通过不依赖蛋白酶体通路的机制实现。