Pathogen Molecular Biology Unit, Department of Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine, University of London, Keppel St, London WC1E 7HT, UK.
J Inflamm (Lond). 2009 Jul 30;6:22. doi: 10.1186/1476-9255-6-22.
Herpesviruses have evolved chemokines and chemokine receptors, which modulate the recruitment of human leukocytes during the inflammatory response to infection. Early post-infection, human herpesvirus 6A (HHV-6A) infected cells express the chemokine receptor U51A and chemokine U83A which have complementary effects in subverting the CC-chemokine family thereby controlling anti-viral leukocyte recruitment. Here we show that, to potentiate this activity, the viral chemokine can also avoid clearance by scavenger chemokine receptors, DARC and D6, which normally regulate an inflammatory response. Conversely, U83A delays internalisation of its signalling target receptor CCR5 with diversion to caveolin rich membrane domains. This mechanism can redirect displaced human chemokines to DARC and D6 for clearance of the anti-viral inflammatory response, leaving the viral chemokine unchecked.
Cell models for competitive binding assays were established using radiolabeled human chemokines and cold U83A on CCR5, DARC or D6 expressing cells. Flow cytometry was used to assess specific chemotaxis of CCR5 bearing cells to U83A, and internalisation of CCR5 specific chemokine CCL4 after stimulation with U83A. Internalisation analyses were supported by confocal microscopy of internalisation and co-localisation of CCR5 with caveosome marker caveolin-1, after virus or human chemokine stimulation.
U83A displaced efficiently human chemokines from CCR5, with a high affinity of 0.01nM, but not from DARC or D6. Signalling via CCR5 resulted in specific chemoattraction of primary human leukocytes bearing CCR5. However, U83A effective binding and signalling to CCR5 resulted in delayed internalisation and recycling up to 2 hours in the absence of continual re-stimulation. This resulted in diversion to a delayed caveolin-linked pathway rather than the rapid clathrin mediated endocytosis previously shown with human chemokines CCL3 or CCL4.
U83A diverts human chemokines from signalling, but not regulatory or scavenger, receptors facilitating their clearance, while occupying signalling receptors at the cell surface. This can enhance virus specific inflammation, facilitating dissemination to replication sensitive leukocytes while evading clearance; this has implications for linked neuro-inflammatory pathologies.
疱疹病毒进化出了趋化因子和趋化因子受体，它们在感染后的炎症反应中调节人类白细胞的募集。在感染早期，人类疱疹病毒 6A（HHV-6A）感染的细胞表达趋化因子受体 U51A 和趋化因子 U83A，它们在颠覆 CC 趋化因子家族方面具有互补作用，从而控制抗病毒白细胞的募集。在这里，我们表明，为了增强这种活性，病毒趋化因子还可以避免被清道夫趋化因子受体 DARC 和 D6 清除，而 DARC 和 D6 通常可以调节炎症反应。相反，U83A 延迟了其信号靶受体 CCR5 的内化，并将其转移到富含 caveolin 的膜域。这种机制可以将移位的人趋化因子转移到 DARC 和 D6 上，以清除抗病毒炎症反应，而不会使病毒趋化因子失控。
使用放射性标记的人趋化因子和 U83A 在表达 CCR5、DARC 或 D6 的细胞上建立竞争结合测定的细胞模型。使用流式细胞术评估 U83A 对携带 CCR5 的细胞的特异性趋化性，以及 U83A 刺激后 CCR5 特异性趋化因子 CCL4 的内化。在病毒或人趋化因子刺激后，通过共聚焦显微镜对 CCR5 与 caveosome 标记物 caveolin-1 的内化和共定位进行了内化分析。
U83A 有效地将人趋化因子从 CCR5 中置换出来，亲和力为 0.01nM，但不能从 DARC 或 D6 中置换出来。通过 CCR5 信号转导导致携带 CCR5 的原代人白细胞的特异性趋化性。然而，U83A 对 CCR5 的有效结合和信号转导导致内化和再循环延迟长达 2 小时，而无需持续再刺激。这导致转向延迟的 caveolin 相关途径，而不是先前用人类趋化因子 CCL3 或 CCL4 所示的快速网格蛋白介导的内吞作用。
U83A 将人趋化因子从信号转导中转移出来，但不转移到调节或清道夫受体中，从而促进其清除，同时占据细胞表面的信号转导受体。这可以增强病毒特异性炎症，促进传播到复制敏感的白细胞，同时逃避清除；这对相关的神经炎症病理学有影响。
