Virus on the screen
3D video surveillance in real time captures the behavior of the virus at an early stage of infection
Tatiana Sashina, PCR.news
One of the first lines of defense of the body against viral infection are layers of closely adjacent epithelial cells lining the respiratory tract and intestines and covered with mucus. The study of how viruses manage to overcome this protection seems to be a non-trivial task. The earliest moments of interaction between the virus and the cell, occurring even before infection, are difficult to trace using existing microscopy methods.
The existing methods of tracking single particles (single-particle tracking, SPT) allowed us to describe the mechanisms of interaction of the virion with the cell surface. However, it was not possible to trace the behavior of virions in the extracellular matrix with a sufficient degree of visualization, since they are immersed too deeply and move too fast for SRT.
Further complicating the visualization task is the fact that viruses are hundreds of times smaller than the cells they infect. "Under a microscope, it's like trying to photograph a person standing in front of a skyscraper. You can't get the whole skyscraper and see the person in front of it in one picture," explains Courtney Johnson, a member of the Duke University team (USA), who developed a way to record the behavior of the virus when approaching the target cell in 3D video format in real time. The method was called 3D-TrIm. It allows you to track the trajectory of a single viral particle against the background of a three-dimensional image of a layer of living cells. The work is published in Nature Methods.
3D-TrIm combines two "microscopes" in one. The first "microscope", implemented on the basis of the technology of three-dimensional active tracking of individual molecules in real time (3D-SMART), is fixed on a fast-moving virus, passing a laser around it tens of thousands of times per second to calculate and update its position. The virus particle must be associated with a fluorescent label. While the virus is bouncing and tumbling in the extracellular matrix, the system is constantly adjusting to keep it in focus. Currently, scientists can only track the virus for a few minutes, then the tag disappears.
The second "microscope" uses the technology of rapid three-dimensional capture with vertical focal scanning (3D-FASTR) to provide a three-dimensional image of the cells around the tracked viral particle. 3D-FASTR is based on two-photon laser scanning microscopy. The microscope must be equipped with an electrically adjustable lens that performs remote laser focusing during continuous vertical focal scanning in the range of 8 microns. The three-dimensional image is formed without moving the sample table and perfectly complements the 3D-SMART.
Using 3D-TrIm, the scientists analyzed the journey of lentiviral particles pseudotyped with vesicular stomatitis virus (VSV-G) G-protein and carrying fluorescent proteins in HeLa or GM701 cell monolayers. Various cell structures were labeled with fluorescent dyes.
The scientists investigated the trajectories of short temporary contacts (skimming) of virus-like particles with the cell surface, and also observed individual cases of longer contacts. Unlike skimming, they were accompanied by sharp changes in the ability of particles to diffuse in the matrix. The virus first undergoes several skimming events, and after about 70 seconds, when the distance between the virus and the cell reaches a minimum, the diffusion capacity of the particle drops by two orders of magnitude and it remains bound to the surface. The scientists also noticed that the bound viral particles can separate from the cell surface, move further in the matrix, or even be adsorbed again elsewhere.
The 3D-TrIm method allows you to track nanoscale structures on the cell surface. So, the scientists analyzed the interaction between VLP and cylindrical protrusions on the cell membrane. Despite the fact that the protrusion is not colored and, therefore, should not be visible in the image, the path of the virus along its surface creates a high-resolution map, as if cutting out the nanoscale cylindrical morphology of the surface of the protrusion in space.
The authors of the work also demonstrated the great potential of 3D-TrIm for the analysis of complex multilayer cellular models that better reproduce in vivo conditions. For example, it is possible to study the extracellular dynamics of viruses (including respiratory viruses) on a system that includes an epithelium protected by a thick layer of mucus and a periciliary layer. The authors created such a system by growing colon epithelial cells on a semipermeable membrane substrate. The substrate was turned over so that the cells hung above the surface of the cover glass. In an experiment with a pseudovirus, scientists found significant, previously undescribed changes in the dynamics of the virion when it interacts with the cell surface in this system.
The authors note that 3D-TrIm is fully compatible with active biological processes, which makes it possible to study other stages of the virus life cycle occurring inside a living cell. It is important to note that this method can be used in any system where the rapid dynamics of nanoscale objects occurs, including, for example, the delivery of nanoscale candidate drugs to the lungs and through the vascular network of the tumor.
Article by Johnson et al. Capturing the start point of the virus–cell interaction with high-speed 3D single-virus tracking is published in the journal Nature Methods.
The video shows how the virus (purple path) finds its way to the surface of human intestinal cells (green).
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