Unlike transport vesicles or organelles, human adenovirus (HAdV) directly binds to the microtubule minus end-directed motor dynein for transport to the nucleus. The
dynein cofactor dynactin enhances nuclear transport of HAdV and boosts infection. To determine if dynactin has a specific role in cytoplasmic trafficking of incoming HAdV on
microtubules, we used live cell spinning disc confocal microscopy at 25 Hz acquisition frequency and automated tracking of single virus particles at 20–50 nm spatial resolution.
Computational dissection by machine-learning algorithms extracted specific motion patterns of viral trajectories. We found that unperturbed cells supported two kinds of
microtubule-dependent motions, directed motions (DM) and fast drifts (FD). DM had speeds of 0.2 to 2 μm/s and run lengths of 0.4 up to 7 μm, while FD were slower and less
extensive at 0.02 to 0.4 μm/s and 0.05 to 2.5 μm. Dynactin interference by overexpression of p50/dynamitin or a coiled-coil domain of p150/Glued reduced the speeds and amounts of both center- and periphery-directed DM but not FD, and inhibited infection. These results indicate that dynactin enhances adenovirus infection by increasing the speed and efficiency of dynein-mediated virus motion to the nucleus, and, surprisingly, also supports a hereto unknown motor activity for virus transport to the cell periphery