Structural and functional characterization of the chemokine enhancing activity ofvaricella zoster virus (VZV) glycoprotein C

水痘带状疱疹病毒（VZV）是一种高度流行的人类病原体，在周围神经元中建立潜伏期。VZV在初次感染时引起水痘，再激活后引起带状疱疹。带状疱疹的特征是急性疼痛，随后可能出现带状疱疹后神经痛（PHN），这是世界上第二常见的神经性疼痛类型。尽管已经有许可的疫苗，但与VZV相关的疼痛仍然是一个主要的健康问题，造成了重要的经济负担。此外，VZV在感染中枢神经系统时还可引起与高死亡率和发病率相关的病理，如脑炎和血管炎。在自然感染期间，VZV有效地感染白细胞，这种感染事件对于VZV在整个宿主中传播到皮肤和神经元以及随后的发病至关重要。白细胞向感染部位的迁移是由趋化因子策划的，一些病毒表达病毒趋化因子结合蛋白（vCKBP）来调节趋化因子的活性。我们最近表明，VZV糖蛋白C （gC）是一种vCKBP，它可以增强趋化因子活性，从而增加白细胞迁移，这一表型可能有助于VZV的传播，与大多数抑制白细胞迁移的vCKBP相反。晶体结构已被报道与趋化因子复合物抑制vCKBP，但没有这样的结构信息可用于增强vCKBP。最后，趋化因子和病毒蛋白与趋化因子/趋化因子受体对相互作用诱导疼痛。因此，鉴于其趋化因子增强活性，可以合理地假设gC在VZV相关疼痛中起重要作用。该项目的目的是进一步研究VZV gC在病毒发病机制中的作用，特别是在VZV相关疼痛的发展中。为此，我们将采用蛋白质生物化学和细胞生物学相结合的方法。我们将首先开始结晶最小功能活性可溶性gC外畴单独和与趋化因子的复合物，并确定其x射线结构。由于糖蛋白往往难以结晶，而抗体片段往往在这种困难的情况下有利于结晶（即作为“结晶伴侣”），我们将生成针对gC的抗体文库。同时，我们将通过分析gC对趋化因子受体使用、聚类和信号传导的影响来确定gC的机制作用。最后，我们将使用动物模型和靶向gC-趋化因子相互作用的α-gC抗体作为特异性对照，研究gC在趋化因子介导的疼痛发展中的作用。我们的研究结果将有助于理解抑制和增强vCKBP之间的差异，确定gC在VZV发病机制中的作用，并为抑制VZV传播和对抗VZV相关疼痛的新治疗方法铺平道路。

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in peripheral neurons. VZV causes varicella during primary infection and zoster following reactivation. Zoster is characterized by acute pain and may be followed by post herpetic neuralgia (PHN), the second most common type of neuropathic pain worldwide. Despite the existence of licensed vaccines, VZV associated pain still represents a major health problem that causes an important economic burden. Moreover, VZV can also cause pathologies associated with high mortality and morbidity such as encephalitis and vasculitis when infecting the central nervous system.During natural infection VZV productively infects leukocytes and this infection event is essential for VZV spread throughout the host to the skin and neurons and also for subsequent pathogenesis. Leukocyte migration to the site of infection is orchestrated by chemokines and several viruses express viral chemokine binding proteins (vCKBP) to modulate chemokine activity. We have recently shown that VZV glycoprotein C (gC) is a vCKBP that enhances chemokine activity resulting in increased leukocyte migration - a phenotype that likely contributes to VZV spread, in contrast to the majority of vCKBP that inhibit leukocyte migration. Crystal structures have been reported for inhibitory vCKBP in complex with chemokines, but no such structural information is available for enhancing vCKBP. Finally, chemokines and viral proteins that interact with the chemokine/chemokine receptor pair induce pain. Therefore, in view of its chemokine-enhancing activity it is plausible to assume that gC plays an important role in VZV associated pain.The goal of this project is to further investigate the role of VZV gC in viral pathogenesis and in particular in the development of VZV associated pain. For this purpose, we will employ a combination of protein biochemistry and cell biology approaches. We will first set out to crystallize the minimal functionally-active soluble gC ectodomain alone and in complex with a chemokine and determine its X-ray structure(s). As glycoproteins are often difficult to crystallize and antibody fragments often facilitate crystallization in such difficult cases (i.e., they serve as "crystallization chaperones"), we will generate an antibody library targeting gC. In parallel, we will determine the mechanistic role of gC by analyzing its impact on chemokine receptor usage, clustering and signalling. Eventually, we will investigate the role of gC in the development of chemokine-mediated pain using an animal model and α-gC antibodies targeting the gC-chemokine interaction as specific control. Our results will contribute to understanding of the differences between inhibitory and enhancing vCKBP, to determine the role of gC in VZV pathogenesis and pave the way for novel therapeutic approaches to inhibit VZV spread and to combat VZV associated pain.