Herpes simplex virus type 1 immediate-early polypeptide Vmw110 (ICP0) is a general transactivator of gene expression in transfection assays and is required for the fully efficient onset of viral lytic replication. It has also been implicated in the process of viral reactivation from latency. Its mechanism of action is unknown, but any involvement in latency requires interactions between viral and host factors. We have previously shown that Vmw110 binds to a 135-kDa cellular protein. In this paper we define a short region towards the C-terminal end of Vmw110 that is required for the 135-kDa protein interaction in virus-infected cells and in vitro. We also confirm that the C-terminal region of Vmw110 contains residues that are responsible for the multimerisation of the protein; these sequences are at least partially distinct from those involved in 135-kDa binding. Both multimerisation and 135-kDa protein interaction are required for full viral infectivity, and elimination of these functions affects the normal interactions between Vmw110 and cellular nuclear structures that contain the PML protein.