Our studies have focused on natural variants and gene deletion mutants of herpes simplex virus type 1 (HSV-1) in order to identify viral proteins important for neuronal transport and assembly. We have examined HSV-1 assembly in the distal axon and growth cone, using a unique technique of longitudinal sectioning of human and rat dorsal root ganglionic neurones and transmission electron microscopy to identify virions assembling within the distal axon. On this basis we have identified a major role for the HSV-1 envelope protein pUS9 in HSV-1 assembly in growth cones at the axon terminus but not in the cell body of neurones. This was observed for a specific pUS9 deletion virus which had a major assembly defect in the growth cone, which was reversed in a repair virus. This is supported by similar observations with the natural HSV-1 KOS strain which is known not to express pUS9. We have also shown the microtubule-dependent cellular molecular motor kinesin-1 to be present by transmission immunoelectron microscopy on HSV-1 capsids, as well as on enveloped virions, in axons. pUS9 is also a candidate for a role in anterograde axonal transport of HSV-1 at its initiation through loading onto kinesin-1 in the cell body. In support of this we have identified a kinesin-1 binding domain in pUS9.