HSV-1 virions contain a proteinaceous layer termed the tegument that lies between the nucleocapsid and viral envelope. The molecular mechanisms that facilitate incorporation of tegument proteins are poorly characterized. The tegument protein VP22 interacts with VP16 and the cytoplasmic tail of glycoprotein E (gE). Virion incorporation of VP22 occurs independently of interaction with VP16; however, the contribution of gE binding remains undefined. Site-directed mutagenesis was used to identify VP22 mutants which abrogate interaction with gE but retain VP16 binding. Virion incorporation assays demonstrated that failure to bind gE did not abrogate VP22 packaging. A region of VP22 which binds to both VP16 and gE failed to be packaged efficiently, with wild-type levels of incorporation only attained when residues 43-86 of VP22 were present. Mutational analysis of an acidic cluster of amino acids within this region indicates that this motif facilitates trans-Golgi network (TGN) localization and optimal virion incorporation of VP22.