Adenovirus interacts with minus-end dynein and a plus-end directed factor to traffic along cytoplasmic microtubules [118]. entry process than can be achieved by any single method alone. instead of the host membrane and conduct experiments in the opposite configuration. Here, binding and fusion is studied by monitoring liposomes decorated with host cell receptors interacting with the planar virus-like bilayer containing embedded viral proteins [59C61]. Such an arrangement could be used for screening applications of antivirals that target entry processes, without the need for live virus or pseudotyped particles. In summary, biomimetic platforms enable a level of environmental control that cannot be attained in live cell particle tracking techniques. First, Exatecan Mesylate there is a degree of control over the host cell membrane mimics composition that is difficult to alter in live cells. Second, in these platforms, the buffers in contact with the virus can have a defined composition and the experimenter controls the timing and order of exposure to proteases, pH, or any other component of interest to the virus. But perhaps the most salient feature of this experimental approach is that these platforms allow detailed examination of the binding and membrane Exatecan Mesylate fusion process and Mouse monoclonal to NME1 gathering of dynamic data from these processes. However, the two-dimensional, in vitro nature of these platforms make them unsuitable for measuring cytoskeletal involvement in entry. Thus, to obtain the most complete information about the infection process, combining data from complementary approaches using live cells and biomimetic platforms is an excellent strategy. Applications of Single Virion Tracking and Complementary Ensemble Approaches In the following sections we describe how single virion tracking has been applied to investigate different steps in virus entry. We also include overviews of a selection of ensemble methods to appreciate the synergy between the data collected by the different techniques in providing a complete description of virus entry. Table 2.1 provides a quick reference of techniques and the data that can be obtained in each approach for each entry stage. Table 2.1 Comparison of single virion and ensemble methods for studying particular viral entry steps, including key features of each method Quartz crystal microbalance with dissipation, Enzyme-linked immunosorbent assay, Surface plasmon resonance, Transmission electron microscopy,immunofluoresence assay, Beta lactamase Tracking Extracellular Movement of Virions There are two scales of transport to be observed during virus spread and infection. On the larger scale is the transport and spread of virions between neighboring cells. Also of interest is the?smaller-scale tracking of an individual virion on a cell plasma surface before it is internalized by that particular cell. In the following sections, experiments at each scale will be described with selected examples and references. Tracking Virion Movement Between Cells Tracking virion movement in the in vivo environment has revealed various avenues of virus spread to surrounding cells. The predominant transport mechanisms of virus spread between cells are: (1) virions freely diffusing through the extracellular environment to neighboring cell surfaces, or (2) spreading to neighboring cells through direct transmission across adjoining membranes. Exatecan Mesylate For the first mechanism, the mean-squared displacement of virions over time is used to classify their motion as diffusive or sub-diffusive through the extracellular environment. For example, live cell single virion tracking of adeno-associated viruses [17] and simian virus 40 virus-like particles [62] indicates that particles undergo normal diffusion in the extracellular environment. Adeno-associated viruses slow down when in the vicinity of a cell, and touch the cell membrane multiple times before penetrating the cell [17]. In contrast, HIV follows the second mechanism and preferentially transmits directly from one neighboring cell to another through virological synapses rather than transmission by extracellular diffusion [63C65] Some viruses exploit cytoskeletal components to facilitate transport from one cell to another. Vaccinia virus, for example, induces the formation of actin protrusions from the cell surface and is transported along these to spread from cell to cell [66]. Looking at viral transport over a longer distance scale, single virion imaging has shown that pseudorabies virus is able to spread from the site of infection to the peripheral nervous system ganglia for replication then back along axons to reseed the initial infection site [67]. In?vitro experiments have been used to study how respiratory mucosa hinders the ability of pseudorabies virus to cross into the epithelium, revealing that both size and charge interactions are important [68]. Tracking Virion Movement on Live Cell Plasma Membranes There are multiple strategies for observing lateral viral movement.