Transport Modes of Viral Nucleoproteins in Live Cells

Abstract

Within a few hours after infection of a cell by vesicular stomatitis virus (VSV), newly assembled VSV particles are released from the surface of the infected cell. In that time, the viral RNA and 5 viral proteins have travelled to the edge of the cell from the sites of synthesis near the nucleus, a radial distance of 5-10 μm. The modes of transport from the sites of synthesis to the edge of the cell are key questions. The movement of VSV ribonucleoprotein particles (nucleocapsids) in live A549 cells was recorded by fluorescence video microscopy at 100fps at 3 to 4h postinfection. Each nucleocapsid contains approximately 400 molecules of GFP-tagged P protein. Nucleocapsids are tracked to subpixel precision. About 60% of the tracks show spatially constrained, ATP-enhanced Brownian motion. These particles jiggle within an approximately circular area (“trap”) with 2s radii = 0.18 ± 0.05 μm. Particles stay in one trap for 1-5 s, then move abruptly to an adjacent trap. Motion within a trap is not directional. 1-3% of tracks show short bouts (< 1s) of directed motion between traps. The velocity during directed motion is similar in magnitude to the frame-to-frame velocity in the traps. Bayesian analyses based on 2D location, particle velocity, and directional change are being used to identify the traps and the periods of directed motion. Simulations, biochemical perturbation (motor protein inhibitors) and colocalization of capsids with cytoskeletal fibers are being used to discern the physical basis for the observed modes of travel. Supported by grant 1R01 AI120623-01A1 from the US National Institutes of Health.