An-An Liu, Zhenfeng Zhang, En-Ze Sun, Zhenhua Zheng, Zhi-Ling Zhang, Qinxue Hu, Hanzhong Wang, and Dai-Wen Pang
ACS Nano, 2016, 10 (1), pp 1147–1155
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.