| Viral disease, especially the infectious viral disease, has seriously endangered the life safety of human beings and the orderly progress of social activities. Understanding the infection and pathogenic mechanism of virus is significant for developing the prevention and treatment drugs for viral disease. Single virus tracking is an imaging method which is focused on tracking the critical steps of virus infection process in real-time to explore the complicated dynamic interaction between single virus particle and host cell, and finally reveal the mechanism of virus infection. However, the traditional means that using the fluorescent dyes and fluorescent proteins to label virus can hardly realize the long-term tracking of single virus because the weak fluorescence and photobleaching of these fluorescent tag. Using inorganic nanomaterials quantum dots (QDs), which have excellent luminescence properties, can easily monitor the infection path and behaviors of single virus for long time, giving further insight into the virus infection mechanism. In this contribution, we focused on the efficient QDs labelling methods for enveloped virus and the real-time imaging analysis of the dynamic delivery process of virus in living cells. The two main contents of this research are as follows:1. Efficient QDs labelling of respiratory syncytial virusesIn situ virus labelling based on the viral assembly process in His-tag modified cells. Our labelling strategy was based on the fact that when virus assembly and release from the host cell membrane, a large number of viral membrane proteins would exist on the cell surface. After the activation by EDC/NHS, His-tag could be covalently linked to the viral protein on cell surface, which can realize the labelling of His-tag to virus after virus budding from the cells. Then, the QDs-labelling virus could be obtained by binding these viruses and NTA-Ni modified QDs through the chelation of Ni ions and His-tag. The fluorescence colocalized assay of immunofluorescence of virus and QDs fluorescence indicated that71.5±4.1%of virions could be labelled by QDs. And the in situ labelled virus have much higher (455times) infectivity than the labelled virus obtained from the direct modification of living virus. Thus, this method is an efficient virus labelling method that is conducive to get high infectivity labelled virus.Dual-labelling virus by employing the biotinylated host-cell membrane protein and nucleic acid stains for tracking the early stage of virus entry. Based in the fact that some host cells’membrane proteins can incorporate into the mature virions during the budding process of enveloped virus, we proposed the strategy to label virus by employing the host cells’membrane proteins. At first, the host cells were biotinylated to introduce biotin molecules to membrane proteins. Then, these cells were infected by virus. When the mature virions assembly and release from the cells membrane, the biotinylated host membrane proteins could be naturally incorporated into the envelope of virus. Furthermore, by adding the nucleic acid stains that can specifically bind the RNA during the biotin labelling process, the internal and external components of virus could be simultaneously labelled. The QDs labelling efficiency could reach83.7±4.6%because the highly-efficient binding of biotin in viral surface and streptavidin-conjugated QDs. Besides, the use of modified host proteins can avoid the occupation of the active sites on viral surface, making the infectivity of QDs-labelled virus remained95.4%. Finally, by real-time imaging the early stage of dual-labelled virus infection, we studied the dynamic process of virus entry and the rapid release of viral nucleic acid.Double quantum dots labelling of enveloped virus based on the viral assembly process in host cells. Due to the wide excitation range and narrow emission peak, simultaneous use of different QDs can hardly bring the spectral overlay, which make QDs the excellent multi-color labelling probe. However, all of the previous virus labelling methods by QDs only used one kind of QDs. For better tracking the internal and external components of virus for long-term, we designed a dual-labelling strategy for simultaneously labelling the viral membrane protein and nucleic acid by two different QDs. To label the viral nucleic acid, a genome probe composed of QDs and a molecular beacon (MB) that can bind to a specific subsequence on viral genome. One of the ends of MB could link QDs and the other end was a quencher that can quench the fluorescence of QDs, making this probe the fluorescent switch that only the viral genome can turn it on. During the virus replication process, this probe was introduced to the cytoplasm of the infected cells by membrane permeating agents to bind the corresponding subsequence. Then, the labelling of viral surface was carried out by biotinylating the viral membrane proteins that embedded in the cell surface and finally incubated with the streptavidin-conjugated QDs. These two labelling procedures both used the assembly and release process of virus in host cells. Compared with other virus labelling methods, this method can simultaneously label the internal and external components of virus with little effect on the infectivity of virus and can be applied to other enveloped virus that budding from the cell surface.2. Real-time tracking of the dynamic delivery of QDs-Iabelled respiratory syncytial virus in living cellsThrough long-term fluorescence monitoring of QDs-labelled virus, the fluorescence colocalized assay of QDs-labelled virus and cellular proteins/organelles, as well as the drug inhibition experiments, the infection process of respiratory syncytial virus to host cells has been carefully studied. Results showed that this virus entry cells through a rapid and efficient process, which included three critical events:1) dynamin-and caveolin-mediated endocytosis,2) actin-dependent transport and RNA release, and3) microtubules-dependent transport and accumulation in endoplasmic reticulum. In addition, this study also indicated that our QDs-labelling strategy was useful to directly investigate the infection pathway without effect on the internalization path of virus.In summary, our researches were focused on the mild and efficient QDs-labelling method for enveloped virus through the replication and assembly process of virus in host cells, and finally studied the infection pathway of virus in living cells by imaging analysis. These in situ labelling strategies avoided directly operating of living virus, greatly reduced the effect of QDs binding to viral infectivity and realized the double QDs labelling for the first time, which are expected to provide new insights to nanomaterials labelling and expand the application of inorganic fluorescent nanomaterials in bioimaging fields. |
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