Three-dimensional single particle tracking of herpesvirus egress and tegument assembly

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Herpesviruses are among the most complex and largest of the clinically relevant viruses. Herpesviruses are extremely widespread in vertebrates and humans and establish life-long infections in their hosts. Infections by the nine known human herpesviruses are associated with many serious diseases. Although many studies exist about the replication cycle of herpesviruses, not much is known about the later stages of the infection cycle - the assembly of virus particles and their egress from the cell. Support for the accepted model of herpesvirus assembly and egress was provided mostly by electron micrographs. A problem of electron microscopy is the limitation to fixed specimens and non-dynamic states. The goal of this project is to gain new insight and refine the model of herpesvirus assembly and egress by the use of three-dimensional single particle tracking of fluorescently labeled viruses in real time inside living cells. Single-particle tracking is a powerful tool to study the dynamics inside heterogeneous systems like cells because this technique locates each particle with sub-diffraction resolution and measures its individual dynamics, instead of an ensemble average. Compared to previous work reporting on herpesviruses trajectories in cells, the increased observation times afforded by use of active 3D tracking will provide us with much longer trajectories and enable us for the first time to follow the path of the virus particles on timescales of tens of seconds or even minutes. This novel information will be used for the statistical analysis of complex mobility patterns based on individual viruses, identification of dwell times in mobility states and comparison of individual particle behavior to the ensemble average. In the context of cellular compartments, this technique will allow us to observe and elucidate details of viral - cell interactions during processes such as nuclear egress at the nuclear envelope, active transport in the cytoplasm and secondary envelopment at trans-Golgi membranes for example, with high temporal and spatial resolution in real time. Finally, we will combine three-dimensional single particle tracking with dual colour imaging to uncover interactions between virus capsids and viral tegument proteins during virus egress and assembly. By implementation of two-color, three-dimensional single particle tracking we will take the technical capabilities to the limits and open up a completely new way to study herpesvirus protein interactions inside infected cells. Correct tegument assembly is necessary for functional virus particle formation, and thus for successful spreading of virus particles between cells and to new hosts. Live-cell data of tegument assembly is missing and further understanding is of biomedical interest, since it will provide fresh insights into herpesvirus replication, which is important in the search for novel antiviral therapies.