Respiratory syncytial virus fusion protein-induced toll-like receptor 4 (TLR4) signaling is inhibited by the TLR4 antagonists Rhodobacter sphaeroides lipopolysaccharide and eritoran (E5564) and requires direct interaction with MD-2.
Respiratory syncytial virus fusion protein-induced toll-like receptor 4 (TLR4) signaling is inhibited by the TLR4 antagonists Rhodobacter sphaeroides lipopolysaccharide and eritoran (E5564) and requires direct interaction with MD-2.
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作者：
Rallabhandi P;Phillips RL;Boukhvalova MS;Pletneva LM;Shirey KA;Gioannini TL;Weiss JP;Chow JC;Hawkins LD;Vogel SN;Blanco JC
呼吸道合胞病毒（RSV）是全球婴儿死亡的主要原因。Toll样受体4（TLR 4）是一种结构多样的微生物相关分子模式的信号受体，以CD 14依赖性方式被RSV融合（F）蛋白和细菌脂多糖（LPS）激活。通过LPS的TLR 4信号传导还需要另外的蛋白MD-2的存在。因此，F蛋白介导的TLR 4激活也可能依赖于MD-2，尽管这一假设尚未得到正式验证。在表达野生型（WT）TLR 4和CD 14的HEK 293 T转染子中，发现无LPS RSV F蛋白可激活NF-κB，但仅在MD-2共表达时激活。通过测量WT与MD-2−/−巨噬细胞中F蛋白诱导的白细胞介素1β（IL-1β）mRNA证实了这些发现，与WT相比，MD-2−/−巨噬细胞在F蛋白处理后未能显示IL-1β表达。球形红杆菌LPS和合成E5564（eritoran）（通过结合MD-2中的疏水口袋抑制TLR 4信号传导的LPS拮抗剂）均以剂量依赖性方式显著降低HEK 293 T-TLR 4-CD 14-MD-2转染子中RSV F蛋白介导的TLR 4活性，而肿瘤坏死因子α（TNF-α）对TLR 4非依赖性NF-κB活化不受影响。体外免疫共沉淀研究证实了天然RSV F蛋白和MD-2之间的物理相互作用。此外，我们证明了RSV F蛋白的F1片段的N-末端结构域与MD-2相互作用。这些数据为MD-2在RSV F蛋白介导的TLR 4活化中的重要性提供了新的见解。因此，靶向MD-2和RSV F蛋白之间的相互作用可能会导致新的治疗方法，以帮助控制RSV诱导的炎症和病理。这项研究首次表明，呼吸道合胞病毒（RSV）的融合（F）蛋白是毛细支气管炎和死亡的主要原因，特别是在婴儿和幼儿中，通过其N-末端结构域与Toll样受体4（TLR 4）辅助受体MD-2发生物理相互作用。我们发现，F蛋白诱导的TLR 4激活可以被脂质A类似物拮抗剂阻断。这一观察结果为在RSV感染的动物模型中测试此类拮抗剂以用于人的潜在用途提供了强有力的实验原理。
Respiratory syncytial virus (RSV) is a leading cause of infant mortality worldwide. Toll-like receptor 4 (TLR4), a signaling receptor for structurally diverse microbe-associated molecular patterns, is activated by the RSV fusion (F) protein and by bacterial lipopolysaccharide (LPS) in a CD14-dependent manner. TLR4 signaling by LPS also requires the presence of an additional protein, MD-2. Thus, it is possible that F protein-mediated TLR4 activation relies on MD-2 as well, although this hypothesis has not been formally tested. LPS-free RSV F protein was found to activate NF-κB in HEK293T transfectants that express wild-type (WT) TLR4 and CD14, but only when MD-2 was coexpressed. These findings were confirmed by measuring F-protein-induced interleukin 1β (IL-1β) mRNA in WT versus MD-2−/− macrophages, where MD-2−/− macrophages failed to show IL-1β expression upon F-protein treatment, in contrast to the WT. Both Rhodobacter sphaeroides LPS and synthetic E5564 (eritoran), LPS antagonists that inhibit TLR4 signaling by binding a hydrophobic pocket in MD-2, significantly reduced RSV F-protein-mediated TLR4 activity in HEK293T-TLR4–CD14–MD-2 transfectants in a dose-dependent manner, while TLR4-independent NF-κB activation by tumor necrosis factor alpha (TNF-α) was unaffected. In vitro coimmunoprecipitation studies confirmed a physical interaction between native RSV F protein and MD-2. Further, we demonstrated that the N-terminal domain of the F1 segment of RSV F protein interacts with MD-2. These data provide new insights into the importance of MD-2 in RSV F-protein-mediated TLR4 activation. Thus, targeting the interaction between MD-2 and RSV F protein may potentially lead to novel therapeutic approaches to help control RSV-induced inflammation and pathology. This study shows for the first time that the fusion (F) protein of respiratory syncytial virus (RSV), a major cause of bronchiolitis and death, particularly in infants and young children, physically interacts with the Toll-like receptor 4 (TLR4) coreceptor, MD-2, through its N-terminal domain. We show that F protein-induced TLR4 activation can be blocked by lipid A analog antagonists. This observation provides a strong experimental rationale for testing such antagonists in animal models of RSV infection for potential use in people.