Anterograde Viral Tracer Herpes Simplex Virus 1 Strain H129 Transports Primarily as Capsids in Cortical Neuron Axons
Anterograde Viral Tracer Herpes Simplex Virus 1 Strain H129 Transports Primarily as Capsids in Cortical Neuron Axons
复制标题
顺行病毒示踪剂单纯疱疹病毒 1 株 H129 主要作为衣壳在皮质神经元轴突中运输
DOI：
10.1128/jvi.01957-19
复制
发表时间：
2020-04-01
影响因子：
5.4
通讯作者：
Zeng, Wen-Bo
中科院分区：
文献类型：
--
作者：
Dong, Xiao;Zhou, Jing;Zeng, Wen-Bo
单纯疱疹病毒1 （HSV-1）株129 （H129）的顺行跨神经元示踪剂是绘制神经回路解剖和功能连接的重要工具。因此，阐明H129在神经元内和神经元间的转运模式至关重要。我们用基因编码的荧光标记衣壳蛋白和/或糖蛋白构建重组H129变体，以观察病毒颗粒在神经元中的运动。电镜和光镜数据显示，H129衣壳和包膜是分开运动的，值得注意的是，衣壳被包裹在轴突静脉曲张和末端，这是形成突触连接其他神经元的部位。基于超分辨率显微镜的共定位分析和分子马达抑制剂对H129颗粒运动的抑制支持了激酶-1参与衣壳的顺行运输。这些结果揭示了h129来源的示踪剂在轴突内的顺行运输和神经元间通过突触的传递机制，解释了h129来源的示踪剂对神经回路的顺行标记。单纯疱疹病毒1 （HSV-1）毒株129 （H129）具有天然的神经亲和性和顺行跨神经元转运的特点，使其成为顺行神经回路追踪的潜在工具。近年来，以H129为基础开发了顺行多突触和单突触示踪剂，并应用于识别不同神经回路的新连接和功能。然而，H129病毒颗粒是如何在神经元，特别是中枢神经系统中运输的，目前尚不清楚。在本研究中，我们构建了带有mccherry标记的衣壳和/或绿色荧光蛋白（GFP）标记的包膜的重组H129变异体，并感染皮质神经元以研究H129病毒颗粒的轴突转运。我们发现不同类型的病毒颗粒不均匀地分布在细胞核、细胞质和轴突中。大多数H129子粒子为无包膜衣壳，在轴突中以衣壳而不是病毒粒子的形式运输。值得注意的是，衣壳在轴突曲张和末端获得包膜，在那里形成突触的位点与其他神经元相连。此外，病毒衣壳在轴突内的顺行运动频率更高，平均速度为0.62±0.18 μm/s，最大速度为1.80±0.15 μm/s。我们还提供了证据，证明衣壳的轴突运输需要动力蛋白-1分子马达。这些发现支持h129衍生的示踪剂可以顺行并可能通过突触映射神经回路。这些数据将指导未来基于h129的顺行病毒示踪剂的修改和改进。从单纯疱疹病毒1 （HSV-1）株129 （H129）衍生的顺行跨神经元示踪剂是绘制神经回路解剖和功能连接的重要工具。因此，阐明H129在神经元内和神经元间的转运模式至关重要。我们用基因编码的荧光标记衣壳蛋白和/或糖蛋白构建重组H129变体，以观察病毒颗粒在神经元中的运动。电镜和光镜数据显示，H129衣壳和包膜是分开运动的，值得注意的是，衣壳被包裹在轴突静脉曲张和末端，这是形成突触连接其他神经元的部位。基于超分辨率显微镜的共定位分析和分子马达抑制剂对H129颗粒运动的抑制支持了激酶-1参与衣壳的顺行运输。这些结果揭示了h129来源的示踪剂在轴突内的顺行运输和神经元间通过突触的传递机制，解释了h129来源的示踪剂对神经回路的顺行标记。
Anterograde transneuronal tracers derived from herpes simplex virus 1 (HSV-1) strain 129 (H129) are important tools for mapping neural circuit anatomic and functional connections. It is, therefore, critical to elucidate the transport pattern of H129 within neurons and between neurons. We constructed recombinant H129 variants with genetically encoded fluorescence-labeled capsid protein and/or glycoprotein to visualize viral particle movement in neurons. Both electron microscopy and light microscopy data show that H129 capsids and envelopes move separately, and notably, capsids are enveloped at axonal varicosity and terminals, which are the sites forming synapses to connect with other neurons. Superresolution microscopy-based colocalization analysis and inhibition of H129 particle movement by inhibitors of molecular motors support that kinesin-1 contributes to the anterograde transport of capsids. These results shed light into the mechanisms for anterograde transport of H129-derived tracer in axons and transmission between neurons via synapses, explaining the anterograde labeling of neural circuits by H129-derived tracers. ABSTRACT The features of herpes simplex virus 1 (HSV-1) strain 129 (H129), including natural neurotropism and anterograde transneuronal trafficking, make it a potential tool for anterograde neural circuitry tracing. Recently anterograde polysynaptic and monosynaptic tracers were developed from H129 and have been applied for the identification of novel connections and functions of different neural circuitries. However, how H129 viral particles are transported in neurons, especially those of the central nervous system, remains unclear. In this study, we constructed recombinant H129 variants with mCherry-labeled capsids and/or green fluorescent protein (GFP)-labeled envelopes and infected the cortical neurons to study axonal transport of H129 viral particles. We found that different types of viral particles were unevenly distributed in the nucleus, cytoplasm of the cell body, and axon. Most H129 progeny particles were unenveloped capsids and were transported as capsids rather than virions in the axon. Notably, capsids acquired envelopes at axonal varicosities and terminals where the sites forming synapses are connected with other neurons. Moreover, viral capsids moved more frequently in the anterograde direction in axons, with an average velocity of 0.62 ± 0.18 μm/s and maximal velocity of 1.80 ± 0.15 μm/s. We also provided evidence that axonal transport of capsids requires the kinesin-1 molecular motor. These findings support that H129-derived tracers map the neural circuit anterogradely and possibly transsynaptically. These data will guide future modifications and improvements of H129-based anterograde viral tracers. IMPORTANCE Anterograde transneuronal tracers derived from herpes simplex virus 1 (HSV-1) strain 129 (H129) are important tools for mapping neural circuit anatomic and functional connections. It is, therefore, critical to elucidate the transport pattern of H129 within neurons and between neurons. We constructed recombinant H129 variants with genetically encoded fluorescence-labeled capsid protein and/or glycoprotein to visualize viral particle movement in neurons. Both electron microscopy and light microscopy data show that H129 capsids and envelopes move separately, and notably, capsids are enveloped at axonal varicosity and terminals, which are the sites forming synapses to connect with other neurons. Superresolution microscopy-based colocalization analysis and inhibition of H129 particle movement by inhibitors of molecular motors support that kinesin-1 contributes to the anterograde transport of capsids. These results shed light into the mechanisms for anterograde transport of H129-derived tracer in axons and transmission between neurons via synapses, explaining the anterograde labeling of neural circuits by H129-derived tracers.