武汉病毒所建立病毒感染细胞示踪新方法

屈泰龙

病毒感染宿主细胞，从入侵到释放这一复制周期中的各个环节是病毒学研究的最基本问题。想要追踪病毒的感染过程，揭示其入侵机制，实现活病毒感染细胞的全程单颗粒病毒示踪是一种重要手段，但建立实时单颗粒病毒的动态示踪，特别是实现新生子代病毒的单颗粒动态示踪新方法是一个极具有挑战性的课题。

　　近日，中科院武汉病毒所王汉中研究员同武汉大学庞代文课题组合作，依据HaloTag（一种标签蛋白）在与不同配体结合时能发出不同颜色的光这一原理，通过反向遗传学技术将HaloTag与伪狂犬病毒（Pseudorabies virus， PrV)衣壳蛋白VP26融合表达，构建了嵌合有Halo Tag的重组PrV，并在PrV感染细胞的不同阶段采用不同的配体对PrV进行标记，根据重组PrV在不同的激发光下所产生颜色的差异区别亲代病毒和子代病毒，完成了从PrV吸附、入侵、病毒衣壳沿着微管运动与入核、子代PrV在核内的组装、出核及在细胞质内的运动轨迹等病毒复制周期的全程单颗粒动态示踪。该论文已在线发表在纳米领域重要杂志ASC Nano上（DOI：10.1021/acsnano.5b06438）。

　　PrV 是一种非常重要的猪病原体，能引起母猪流产、死胎以及呼吸道感染等症状，具有传播速度快、流行范围广、死亡率高等特点，同时与其他α亚科疱疹病毒一样，PrV另一个非常重要的特性是能在宿主的外周神经系统中建立潜伏感染。初始感染宿主之后，首先病毒的囊膜与外周神经系统的轴突末梢融合进入神经细胞，然后病毒衣壳连同部分皮层蛋白一起可沿逆轴突方向通过远距离的传输进入神经细胞的细胞核并建立潜伏感染。在特定条件下病毒被活化，病毒沿着顺轴突方向转运至神经末梢，再次感染上皮组织。因此，PrV不仅是一个研究α疱疹病毒分子生物学的非常有用的模式病毒，而且是研究神经细胞传导示踪的绝好实验对象。

　　武汉病毒所建立的PrV感染细胞的全程单颗粒示踪技术无疑为α疱疹病毒的潜伏感染机制和人神经细胞传导示踪和神经环路的研究提供了重要的技术平台。

Visualization of virus entry and transport in thecytoplasm by parental rPrV_HT

Transport of progeny viral capsids in the cytoplasm

原文链接：Simultaneous Visualization of Parental and Progeny Viruses by a Capsid-Specific HaloTag Labeling Strategy

Abstract

Real-time, long-term, single-particle tracking (SPT) provides us an opportunity to explore the fate of individual viruses toward understanding the mechanisms underlying virus infection, which in turn could lead to the development of therapeutics against viral diseases. However, the research focusing on the virus assembly and egress by SPT remains a challenge because established labeling strategies could neither specifically label progeny viruses nor make them distinguishable from the parental viruses. Herein, we have established a temporally controllable capsid-specific HaloTag labeling strategy based on reverse genetic technology. VP26, the smallest pseudorabies virus (PrV) capsid protein, was fused with HaloTag protein and labeled with the HaloTag ligand during virus replication. The labeled replication-competent recombinant PrV harvested from medium can be applied directly in SPT experiments without further modification. Thus, virus infectivity, which is critical for the visualization and analysis of viral motion, is retained to the largest extent. Moreover, progeny viruses can be distinguished from parental viruses using diverse HaloTag ligands. Consequently, the entire course of virus infection and replication can be visualized continuously, including virus attachment and capsid entry, transportation of capsids to the nucleus along microtubules, docking of capsids on the nucleus, endonuclear assembly of progeny capsids, and the egress of progeny viruses. In combination with SPT, the established strategy represents a versatile means to reveal the mechanisms and dynamic global picture of the life cycle of a virus.

武汉病毒所