African swine fever virus (ASFV) is a large DNA virus that assembles in perinuclear viral factories located close to the microtubule organizing center. In this study, we have investigated the mechanism by which ASFV reaches the cell surface from the site of assembly. Immunofluorescence microscopy revealed that at 16 h postinfection, mature virions were aligned along microtubules. Furthermore, virus movement to the cell periphery was inhibited when microtubules were depolymerized by nocodazole. In addition, ASFV infection resulted in the increased acetylation of microtubules as well as their protection against depolymerization by nocodazole. Immunofluorescence microscopy showed that conventional kinesin was recruited to virus factories and to a large fraction of virus particles in the cytoplasm. Consistent with a role for conventional kinesin during ASFV egress to the cell periphery, overexpression of the cargo-binding domain of the kinesin light chain severely inhibited the movement of particles to the plasma membrane. Based on our observations, we propose that ASFV is recognized as cargo by conventional kinesin and uses this plus-end microtubule motor to move from perinuclear assembly sites to the plasma membrane.Viral replication and assembly often take place in specialized structures located in the nucleus or in the perinuclear cytoplasm. These specialized sites, called viral inclusions or virus factories, provide a focal point for the replication of virus genomes and the recruitment of structural proteins. Recent work has focused on understanding how viruses travel to, and from, these specialized sites during their infectious cycle. One common viral strategy uses microtubules for directed longrange transport of virus components inside cells (44,52,54). Directional movement along microtubules is mediated by motor proteins which hydrolyze ATP to induce the conformational changes necessary to move cargoes along microtubules (30,62).Movement of cellular materials towards the microtubule organizing center (MTOC) (retrograde transport) usually requires the minus-end directed microtubule motor dynein (62). The transport of cargoes by dynein requires association with dynactin, a heterooligomeric complex of at least nine different polypeptides. This has been demonstrated experimentally by the overexpression of the p50-dynamitin subunit of the dynactin complex, which dissociates the dynactin complex and severely affects vesicular trafficking and organelle distribution (10). Importantly, retrograde transport of several unrelated viruses, such as herpes simplex virus type 1 (HSV-1), adenovirus, vaccinia virus (VV), parvovirus, and human immunodeficiency virus type 1 is inhibited when the dynactin complex is dissociated, suggesting that dynein is required for inward transport of these viruses (18,38,43,57,59).The movement along microtubules, from the MTOC towards the plasma membrane (anterograde transport), involves the kinesin family of motor proteins (62). Conventional kinesin is a heterotetramer consisting of two heav...