PDF(Pubmed)
Abstract：
The rabies virus enters the nervous system by interacting with several molecular targets on host cells to modify behavior and trigger receptor-mediated endocytosis of the virion by poorly understood mechanisms. The rabies virus glycoprotein (RVG) interacts with the muscle acetylcholine receptor and the neuronal α4β2 subtype of the nicotinic acetylcholine receptor (nAChR) family by the putative neurotoxin-like motif. Given that the neurotoxin-like motif is highly homologous to the α7 nAChR subtype selective snake toxin α-bungarotoxin (αBTX), other nAChR subtypes are likely involved. The purpose of this study is to determine the activity of the RVG neurotoxin-like motif on nAChR subtypes that are expressed in brain regions involved in rabid animal behavior. nAChRs were expressed in Xenopus laevis oocytes, and two-electrode voltage clamp electrophysiology was used to collect concentration-response data to measure the functional effects. The RVG peptide preferentially and completely inhibits α7 nAChR ACh-induced currents by a competitive antagonist mechanism. Tested heteromeric nAChRs are also inhibited, but to a lesser extent than the α7 subtype. Residues of the RVG peptide with high sequence homology to αBTX and other neurotoxins were substituted with alanine. Altered RVG neurotoxin-like peptides showed that residues phenylalanine 192, arginine 196, and arginine 199 are important determinants of RVG peptide apparent potency on α7 nAChRs, while serine 195 is not. The evaluation of the rabies ectodomain reaffirmed the observations made with the RVG peptide, illustrating a significant inhibitory impact on α7 nAChR with potency in the nanomolar range. In a mammalian cell culture model of neurons, we confirm that the RVG peptide binds preferentially to cells expressing the α7 nAChR. Defining the activity of the RVG peptide on nAChRs expands our understanding of basic mechanisms in host-pathogen interactions that result in neurological disorders.
摘要：
狂犬病病毒通过与宿主细胞上的几种分子靶标相互作用而进入神经系统，以改变行为并通过鲜为人知的机制触发病毒粒子的受体介导的内吞作用。狂犬病病毒糖蛋白（RVG）通过假定的神经毒素样基序与肌肉乙酰胆碱受体和烟碱乙酰胆碱受体（nAChR）家族的神经元α4β2亚型相互作用。鉴于神经毒素样基序与α7nAChR亚型选择性蛇毒素α-银环蛇毒素（αBTX）高度同源，其他nAChR亚型可能涉及。这项研究的目的是确定RVG神经毒素样基序对nAChR亚型的活性，这些亚型在与狂犬病动物行为有关的大脑区域中表达。nAChRs在非洲爪狼卵母细胞中表达，使用双电极电压钳电生理学收集浓度-反应数据来测量功能效应。RVG肽通过竞争性拮抗剂机制优先并完全抑制α7nAChRACh诱导的电流。测试的异聚nAChRs也被抑制，但程度低于α7亚型。用丙氨酸取代与αBTX和其他神经毒素具有高度序列同源性的RVG肽的残基。改变的RVG神经毒素样肽表明，残基苯丙氨酸192，精氨酸196和精氨酸199是RVG肽对α7nAChRs的表观效力的重要决定因素，而丝氨酸195则不是。狂犬病胞外域的评估重申了RVG肽的观察结果，说明对纳摩尔范围内的效力的α7nAChR具有显著的抑制作用。在哺乳动物细胞培养神经元模型中,我们证实RVG肽优先结合表达α7nAChR的细胞。定义RVG肽对nAChRs的活性扩展了我们对导致神经系统疾病的宿主-病原体相互作用的基本机制的理解。
