Quantum Dots-Based Single-Particle Tracking For Studying The Mechanism Of Virus Infection

Many viruses have caused local or global outbreaks of pandemic diseases in history, which have not only threaten human being’s health but also made huge economic losses for the whole social property. A detailed understanding of virus infection mechanism is very meaningful for fighting with viral infection and treating viral diseases. Viral infection is a complex process, involving many steps and interactions between viruses and various cellular components. Therefore, single-particle tracking (SPT) technique, a remarkable tool for studying real-time and in-situ dynamics of biological events in live cells, is badly required for dissecting viral infection mechanism in live cells. The first step for SPT is fluorescent labeling of the viral components. However, most of traditional fluorescent tags used in SPT have low brightness and poor photostability, which could not meet the requirement for long-term SPT tracking, and considerable efforts and time are consumed to acquire sufficient data for statistical analysis. Quantum dots (QDs) possess excellent optical properties, and make it possible for long-term SPT tracking in live cells with a high signal-to noise ratio, which have attracted researchers’attention in biological fields.In this work, we chose avian H9N2influenza virus as a model system to built a set of QDs-based SPT methods for revealing the viral infection mechanism, which is also expected to be broadly used in other biomedicine fields.The main research works are as follows:Developing a general strategy for high-efficiency labeling of single viruses with QDs. By the strong biotin-streptavidin interaction, this method not only could achieve viral envelope labeled with QDs with a labeling efficiency of about97%, but also has less influence on viral infectivity. This strategy possesses good generality, convenience and stability, and makes it possible to globally track individual viruses in live cells for long term. Meanwhile, by labeling the RNA with Syto82, dual-labeled virus could be exploited to further study the viral infection mechanism in details.Building a two-dimensional single-particle tracking method based on QDs labeling. We investigated the feasibility of tracking method on studying the dynamics of biological events by exploring the transport mechanism of wheat germ agglutinin (WGA) in live cells. The results indicate that the endocytic and exocytic processes of WGA are both five-stage, actin-and microtubule-dependent. By two-channel single-particle tracking, we first visualized that QDs-labeled WGA was finally shedding to the extracellular or reversing to the cytoplasm in live cells. This is very meaningful for the application of QDs-based SPT in biological field, and establishes a theoretical basis for lectins used as drug carriers and anticancer drugs.Building a three-dimensional (3D) single-particle tracking method based on QDs labeling. By rescaling3D single-particle image in axial direction and evaluating the radial symmetry center of the rescaled image, we developed a new-type3D SPT method with localization accuracy of～5nm. Without iterative and numerical-fitting steps, this method is always as accuract as3D Gaussian fitting-based method under different conditions, and with two orders of magnitude computation speed. Considerable time can be reduced for processing high volume data in3D SPT. This method is expected to be broadly used in fast high-accuracy SPT field.Dissecting the infection mechanism of influenza virus by QDs-based SPT. By globally tracking individual viruses in live cells, we investageted the five-stage viral infection pathway and the interaction of the viruses and acidic endosomes near the microtubule organizing center. Further, we dissected the diversity and complexity of the microtubule-dependent behaviors and the characteristic the Rab5-and of Rab7-related behaviors of the viruses. The results indicate that the infection of influenza virus is an actin-and microtubule-dependent, Rab5-related dynamic process for RNA releasing in late endosome. This work uncovers several previously unreported mechanisms on virus infection, and may provide a reasonable thinking about antiviral drug design.