The herpes simplex virus type 1 (HSV-1) UL25 gene encodes a minor capsid protein, pUL25, that is essential for packaging the full-length viral genome. Six regions which contain disordered residues have been identified in the high-resolution structure of pUL25. To investigate the significance of these flexible regions, a panel of plasmids was generated encoding mutant proteins, with each member lacking the disordered residues in one of the six regions. In addition, UL25 constructs were produced, which specified proteins that contained missense mutations individually affecting two of the four regions on the surface of pUL25 predicted from evolutionary trace analysis to be important in protein-protein interactions. The impacts of these mutations on viral DNA packaging, virus assembly, and growth were examined. Of the nine mutant proteins analyzed, five failed to complement the growth of a UL25 deletion mutant in Vero cells. These noncomplementing proteins fell into three classes. Proteins in one class did not alter the DNA packaging phenotype of an HSV-1 UL25 deletion mutant, whereas proteins from the other two classes allowed the UL25 null mutant to package full-length viral DNA. Subsequent analysis of the latter classes of mutant proteins demonstrated that one class enabled the null virus to release enveloped virus particles from U2OS cells, whereas the other class prevented egress of mature HSV-1 capsids from the nucleus. These findings reveal a new role for pUL25 in virion assembly, consistent with its flexible structure and location on the capsid.Herpes simplex virus type 1 (HSV-1) has a large, complex virion structure that is composed of four morphologically distinct components. The central core comprises the linear double-stranded DNA genome, which is compactly organized inside an icosahedral capsid containing four different capsid shell proteins. The pUL6 DNA packaging protein forms the portal at the unique vertex. Two additional DNA packaging proteins, pUL25 and pUL17, are found on the capsid, and these proteins are present as heterodimers at multiple sites on the surface, adjacent to the vertices (20,33,35). A protein matrix containing more than 20 different proteins, referred to as the tegument, surrounds the capsid (15). One of the tegument proteins, pUL36, interacts with pUL25 and probably with VP5, the major capsid shell protein (5,19,23), and these associations are likely to be important for anchoring the tegument to the capsid (5). Finally, the outermost layer consists of a lipid membrane embedded with the viral glycoproteins.Viral DNA synthesis and the early stages of virion assembly take place in the nucleus, within replication compartments (8,25). The viral DNA is replicated as head-to-tail concatemers, and cleavage of the viral DNA into unit-length molecules is tightly associated with the packaging of the DNA into a preformed precursor capsid, the procapsid, through the portal. After the viral DNA is encapsidated, the mature C capsid rapidly buds into the inner nuclear membrane and is released i...