Vaccinia virus (VACV) is a large double-stranded DNA virus with a cytoplasmic site of replication. It has a complex life cycle that produces two distinct infectious virion forms, Intracellular Mature Virions (IMVs) and Extracellular Enveloped Virions (EEVs). The host cell microtubule trafficking machinery is hijacked by the virus at three distinct positions of the viral life cycle. After virus entry, the virus cores are transported to pre-nuclear sites where they form viral factories that ultimately produce fully functional and infectious IMVs. A small proportion of IMVs are further transported to sites of wrapping, where they are enveloped by a host-derived double membrane to form Intracellular Enveloped Virions (IEVs). The IEVs are then transported to the cell periphery to facilitate efficient viral spread. The viral proteins A36, F12 and E2 together with the kinesin-1 microtubule motor protein are thought to be involved in IEV egress from the site of wrapping to the cell periphery, although the exact mechanism of movement is unclear. Until recently, A36 was the only known protein to interact with the kinesin-1 motor through kinesin light chain (KLC), but F12 has also been shown to interact with KLC through E2. The precise mechanism of how the IEV interacts with and activates the kinesin-1 motor protein is unclear, and this study explores the interactions of IEV proteins with KLCs in detail, mapping interactions between KLC and A36 or F12/E2. A36, F12 and E2 also show no sequence or predicted structural homology to any other known proteins, and structural studies were performed in an attempt solve their 3D structure. The CRISPR-Cas9 targeted genome editing tool was also utilised to knockout different KLC isoforms in multiple cell lines to assess their contribution to IEV egress as well as cellular trafficking. These studies will provide insight into the mechanisms behind the spatial and temporal control of kinesin motor activity in the cell.