人单纯疱疹病毒1型-H129株的轴突运输机制

神经环路是脑功能的基础，改造嗜神经病毒，发展高效特异的神经环路示踪工具是解析神经环路和脑功能的关键。前期基于HSV-1的H129株(H129)研发了顺向神经环路示踪工具体系：顺向跨多级工具H129-G4，标记高效，适于荧光显微切片断层成像、高分辨率的全脑成像；首个顺向跨单级工具H129-dTK-tdT，能标记目标脑区的直接输出网络，填补了顺向跨单级示踪工具的空白，已广泛应用于神经科学。证实了H129的轴突运输以核衣壳、顺向运输为主，顺向运输与Kinesin-1相关，逆向运输与Dynein相关。但参与轴突运输的病毒蛋白及其机制仍不清楚。因此，本项目将通过免疫共沉淀和蛋白质谱技术筛选和鉴定参与H129轴突运输的病毒蛋白，明确其作用和靶点，揭示H129轴突运输机制；再基于已有的病毒改造平台精准改造运输相关的病毒蛋白，发展更高效特异的顺向示踪工具，为精确解析神经环路、脑联结图谱和脑功能提供新工具。

Neural circuits are the foundation of the brain connectome and functions. Developing efficient and specific neural circuit tracers by modifying neurotropic viruses is the key for identifying novel neural circuits and revealing their functions. We have developed a sets of anterograde neural circuit tracers from herpes simplex virus -1 (HSV-1) strain H129 (H129). Among these tracers, H129-G4 is a multi-synaptic anterograde tracer with very high labeling efficiency and intensity. Its labeling efficiency and intensity are high enough for high-resolution whole brain imaging by fluorescence Micro-optical Sectioning Tomography (fMOST). The H129-dTK-tdT is the first mono-synaptic anterograde transneuronal tracer, which can be used for revealing the direct output networks in the target brain regions (Mol Neurodegener. 2017. 12:38). These H129-derived anterograde tracers have complemented the existing tracers. Both of H129-G4 and H129-dTK-tdT have been applied for identification of novel connections and functions of different neural circuitries (Nat Neurosci. 2017. 20:559；Nat Neurosci. 2017. 20:1680；Nat Commun. 2017. 8:1676；Cell Rep.2018. 22:1734; PNAS. 2019. 116:3799；Nat Neurosci. 2019. 22:1649; Nat Commun. 2020. 11:183.). The next we discovered that most H129 progeny particles transport in a anterograde direction mainly and as unenveloped capsids rather than completely enveloped virions in the axon. The enveloped virions are found only in the axonal terminal and varicosity, which are the sites where synapses are forming and connecting with other neurons. These data collectively suggest that H129 may transmit to connected neurons via synapses, and these H129-derived tracers are transsynaptic tracers. We also provided evidence that axonal transport of capsids requires the kinesin-1 molecular motor (J Virol. 2020. 94:8). However, molecular motor of dynein is involved in H129 axonal transport, and minor retrograde movement driven by dynein is also observed. In addition, axon terminal invasion of H129 can’t be excluded. Both retrograde movement and axon terminal invasion of H129 may cause misleading and dampen the preciseness and efficiency as an anterograde tracer. .In order to improve the current H129-derived anterograde tracers, retrograde axonal movement of H129 viral particle needs to be reduced, and the anterograde transport needs to be enhanced. The mechanism(s) for axonal transport of H129 viral particles is unclear. In this project we will study the interactions of viral proteins-cellular motors that are involved in H129 axonal transport by immunoprecipitation and identify the viral protein candidates by mass spectrometry analysis; then systematically study the roles of the viral protein candidates in H129 directional transport in axon, elucidate the mechanism(s); further modify the viral protein candidates to enhance the anterograde transport and reduce the retrograde movement; finally develop more rigorous, efficient and specific anterograde viral tracer. Our work will eventually provide novel improved circuit tracers, which will contribute in the study of brain functions and brain diseases.