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化学进展 2021, Vol. 33 Issue (1): 13-24 DOI: 10.7536/PC201045 前一篇   后一篇

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单病毒示踪

王志刚1, 刘书琳1, 刘安安1, 张利娟2, 余聪2, 庞代文1,*()   

  1. 1 南开大学药物化学生物学国家重点实验室 化学学院 分析科学研究中心 医学院 天津市生物传感及分子识别重点实验室 天津 300071
    2 武汉大学化学与分子科学学院 分析科学研究中心 武汉 430072
  • 收稿日期:2020-10-27 修回日期:2020-11-18 出版日期:2021-01-24 发布日期:2020-12-09
  • 通讯作者: 庞代文
  • 作者简介:
    * Corresponding author e-mail:
  • 基金资助:
    国家自然科学基金项目资助,纪念南开大学化学学科创建100周年(21877102); 国家自然科学基金项目资助,纪念南开大学化学学科创建100周年(21977054); 国家自然科学基金项目资助,纪念南开大学化学学科创建100周年(91859123); 国家自然科学基金项目资助,纪念南开大学化学学科创建100周年(91953107); 纪念南开大学化学学科创建100周年
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Single-Virus Tracking

Zhi-Gang Wang1, Shu-Lin Liu1, An-An Liu1, Li-Juan Zhang2, Cong Yu2, Dai-Wen Pang1,*()   

  1. 1 Key Laboratory of Medicinal Chemistry Biology, College of Chemistry, Research Center for Analytical Sciences, School of Medicine, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Nankai University, Tianjin 300071, China
    2 College of Chemistry and Molecular Sciences, Research Center of Analytical Sciences, Wuhan University,Wuhan 430072, China
  • Received:2020-10-27 Revised:2020-11-18 Online:2021-01-24 Published:2020-12-09
  • Contact: Dai-Wen Pang
  • Supported by:
    the National Natural Science Foundation of China(21877102); the National Natural Science Foundation of China(21977054); the National Natural Science Foundation of China(91859123); the National Natural Science Foundation of China(91953107); Dedicated to the 100th anniversary of Chemistry at Nankai University

病毒是对人类健康威胁最大的病原之一,由其引发的病毒性疾病对人民健康、国家安全和社会经济构成重大威胁。病毒感染机制研究对病毒性疾病的防控及治疗具有重大意义。病毒侵染宿主细胞的动态过程涉及病毒组分与多种细胞组分或细胞器间复杂的相互作用,但是传统手段无法对该动态过程进行实时跟踪研究。单病毒示踪技术作为一种可以实时原位示踪单颗病毒侵染过程的技术,在病毒感染机制研究方面起着愈来愈重要的作用。借助于该技术,能够获取病毒侵染过程中病毒入胞途径、胞内转运过程、病毒核酸释放及其与细胞间的相互作用等动态过程信息,从而对病毒的感染机制进行分子水平的深入研究。本文就单病毒示踪技术所涉及的测量技术、标记技术和信息获取进行阐述,总结归纳了单病毒示踪在病毒感染机制研究中的最新进展,并探讨了该技术目前存在的挑战性问题以及未来发展的方向。

关键词： 病毒, 示踪, 测量, 标记, 感染, 机制

Viruses are one of the biggest threats to human health, and the outbreak of viral diseases not only poses a great threat to human health and national security, but also causes great losses to the social economy. Uncovering the mechanisms of virus infection is crucial for preventing the spread of viruses and treating viral diseases. The dynamic process of virus infection in host cells involves intricate interactions between viral components and cellular structures or organelles, but conventional methods lack the ability to acquire dynamic information on individual viruses during the infection process. Single-virus tracking(SVT) technique is a powerful approach for studying the real-time and in-situ dynamics of viral processes in live cells and it plays an increasingly important role in the study of viral infection mechanism. SVT allows researchers to obtain the dynamic information on individual viruses during the infection process, including viral entry, trafficking, and genome release, which is meaningful to study the infection mechanisms on the molecular level. In this article, we first discuss the measurement techniques, viral labeling strategies and data analysis methods for SVT, then summarize a couple of applications of SVT and finally propose the challenges and future possibilities of the SVT technique.

Contents

1 Introduction

2 Single-virus tracking technique

2.1 Measurement techniques

2.2 Viral labeling strategies

2.3 Data acquisition

3 Applications of single-virus tracking in virological research

3.1 Virus internalization

3.2 Virus transport

3.3 Genome release of viruses

3.4 Assembly and egress of viruses

4 Challenges and solutions

4.1 Viral labeling strategies

4.2 Measurement techniques

5 Conclusion and outlooks

Key words: virus, tracking, measurement, labeling, infection, mechanism
()
图1 病毒形态多样性 (A)球状(艾滋病毒),(B)杆状(杆状病毒),(C)丝状(埃博拉病毒),(D)弹状(狂犬病毒),(E)砖状(正痘病毒属),(F)冠状(新型冠状病毒),(G)星状(星状病毒),(H)正二十面体(腺病毒)[4 ? ? ? ? ? ? ?~12]
Fig. 1 Various types of virion morphologies. (A) spherical virus(Human immunodeficiency virus),(B) rod-shaped virus(Baculovirus),(C) filamentous virus(Ebol a),(D) bullet-shaped virus(Rabies Virus),(E) brick-shaped virus(Orthopoxvirus),(F) spherical virus with spike protein on the surface(Coronavirus),(G) non-envelope asteroid shaped virus(Astrovirus),(H) icosahedron shaped virus(Adenovirus)[4 ? ? ? ? ? ? ?~12]
图2 单病毒示踪的成像过程。A)病毒颗粒定位示意图。对一个光斑在显微镜上可以获得一个横向分辨率~200 nm,纵向分辨率~500 nm的图像。在二维图像上,光斑强度分布符合二维高斯公式,因此可以采用二维高斯拟合方法对病毒颗粒进行精确定位。B)单病毒示踪包括以下四步:1)图像获取;2)病毒定位;3)轨迹重建;4)轨迹分析。根据MSD和时间间隔的关系,颗粒运动可以分为四类:i)定向扩散运动模式;ii)正常扩散运动模式;iii)非正常扩散运动模式;iv)围栏式扩散运动模式[33]
Fig. 2 Image processing for single-virus tracking.(A) Schematic diagram of particle detection. For one bright particle, the image can be acquired using a fluorescence microscope, which has an ellipsoid shape with ~250 nm in the lateral direction, and ~500 nm in the axial direction. By 2D imaging, the intensity of the particle is more like the 2D Gaussian function. Localization methods are utilized to obtain the accurate position of the particle.(B) Four steps of image processing for single-virus tracking.(1) Recording the particle movements using a microscope in a series of images.(2) Detecting the particle positions in each frame. According to localization algorithms, the accurate particle positions can be acquired.(3) Reconstructing the particle trajectories in the images.(4) Analyzing the trajectories of the particles. According to the relationship between MSD and nΔt, the particle movements can be divided into four types.(i) Directed motion with diffusion.(ii) Normal diffusion.(iii) Anomalous diffusion.(iv) Corralled diffusion[33]
图3 (A)IAV在细胞表面募集网格蛋白和发动蛋白的时间序列图(标尺5 μm);(B)图A方框中三种信号随时间的波动曲线;(C)病毒的运动速度和网格蛋白、发动蛋白的荧光强度曲线;(D)IAV通过依赖网格蛋白和不依赖网格蛋白内吞途径进胞的示意图[29]
Fig. 3 (A) Snapshots of IAV recruiting clathrin and dynamin on plasma membrane. Scale bar, 5 μm.(B) Kymograph images of the virus, clathrin and dynamin signals in panel A.(C) Time trajectories of virus speed and fluorescence intensity of clathrin and dynamin in panel A.(D) Models of IAV entry into cells via clathrin-dependent and clathrin-independent endocytic pathways[29]
图4 (A)IAV在肌球蛋白VI驱动下沿微丝转运;(B)病毒在动力蛋白驱动下沿微管转运;(C)病毒沿微丝和微管转运示意图[52]
Fig. 4 (A) Myosin VI(MyoVI) driving IAV along microfilaments(MF).(B) Dynein driving IAV along microtubules(MT).(C) Model for IAV moving along microfilaments and microtubules[52]
图5 单病毒示踪研究流感病毒不依赖于Rab5蛋白的细胞自噬转运过程
Fig. 5 Uncovering the Rab5-independent autophagic trafficking of influenza A virus by SVT
图6 (A)用两种颜色的量子点分别标记IAV不同节段的基因;(B和C)QD625(红色)和QD705(绿色)同时标记了基因组和QD525(青色)标记了包膜的病毒在细胞核附近释放基因组(标尺10 μm和2 μm);(D和E)在正常细胞和金刚烷胺处理了的细胞内释放了基因组的病毒量(标尺10 μm)[59]
Fig. 6 (A) Labeling the different genome segments of IAV with different quantum dots.(B and C) A virus particle with QD625(red)- and QD705(green)-labeled genome and QD525(cyan)-labeled envelope releasing its genome near the nucleus. Scale bars, 10 μm in panel B and 2 μm in panel C.(D and E) Percentages of viruses releasing genomes in normal and amantadine-treated cells. Scale bar, 10 μm[59]
图7 (A)可视化全长HIV病毒RNA翻译过程的方法示意图;(B)未翻译的RNA(红色)与Gag蛋白(青色)在细胞质膜相互作用并定位在一起;(C)正在翻译的RNA(黄色)到达细胞质膜并离开;(D)正在翻译的RNA在质膜附近的停留时间
Fig. 7 (A) Schematic diagram for the visualization of the translation of full-length HIV RNA.(B) Nontranslating RNA(red) interacting and being colocalized with Gag(cyan) near the plasma membrane.(C) Translating RNA(yellow) reaching and leaving the plasma membrane.(D) Residence time of the RNA in translation staying near the plasma membrane[60]
图8 (A)用Halo标签分别标记亲代和子代伪狂犬病毒的方法示意图;(B~D)子代病毒核衣壳在细胞核内组装的荧光图片、透射电子显微镜图片及三维荧光图片(标尺10 μm);(E)子代核衣壳在细胞核内的运动行为[60]
Fig. 8 (A) Schematic diagram of labeling parental and progeny PrV with Halo tags.(B~D) The fluorescence image, transmission electron image and three dimensional fluorescence image of progeny capsids in the cell nucleus. Scale bars, 10 μm(E) Speeds and MSD of the arrowed capsid in panel B, and statistical diffusion coefficient of capsids diffusing in cell nuclei[63]
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摘要

单病毒示踪