Late in infection herpesviruses move DNA-filled capsids from the nucleus to the cytoplasm by enveloping DNA-containing capsids at the inner nuclear membrane (INM) and deenveloping them at the outer nuclear membrane. This process requires two conserved herpesvirus proteins, pUL31 and pUL34. Interaction between pUL34 and pUL31 is essential for targeting both proteins to the nuclear envelope (NE), and sequences that mediate the targeting interaction have been mapped in both proteins. Here, we show that a mutation in the INM-targeting domain of pUL34 fails to support production of infectious virus or plaque formation. The mutation results in multiple defects, including impaired interaction between pUL34 and pUL31, poor NE targeting of pUL34, and misregulated, capsid-independent budding of the NE. The mutant defects in virus production, plaque formation, and pUL31 interaction can be suppressed by other mutations in the INMtargeting domain of pUL31 and by additional mutations in the pUL34 coding sequence.Efficient nuclear egress of herpesviruses requires formation of a nuclear envelopment complex (NEC) consisting of viral and cellular proteins (24,26,34). The NEC contains homologs of the herpes simplex virus (HSV) UL31 and UL34 proteins (called pUL31 and pUL34), and these are critical for nuclear egress in all herpesviruses tested (9,15,24,25,33). pUL31 and pUL34 homologs interact with each other, and formation of a pUL31/pUL34 complex is required for proper targeting of the complex to the nuclear envelope (NE) (10,16,30,31,36,37,41). The interactions that underlie complex formation are therefore critical for assembly and egress of all herpesviruses.Despite the conservation of a pUL31-pUL34 interaction, it is not clear that the structural basis for that interaction is completely conserved. The sequence of pUL31 and its homologs can be divided into four conserved regions (CRs) (Fig. 1B) (37). The most N-terminal of these regions (CR1) has been shown to mediate interaction with pUL34 homologs in examples from all herpesvirus subfamilies (19, 37). The situation with pUL34 homologs is less clear. For HSV-1 pUL34, the sequence that interacts with pUL31 and that is required for nuclear envelope targeting was mapped by deletion and domain swapping to amino acids (aa) 137 to 181 (18). This corresponds to the third of three CRs in the pUL34 sequence (Fig. 1A). Consistent with this, a construct containing CR1, CR2, and most of CR3 (aa 1 to 161) of pseudorabies virus (PRV) pUL34 was sufficient to interact with pUL31 in a yeast two-hybrid assay (10). In mouse cytomegalovirus (MCMV), on the other hand, use of small insertions and point mutations implicated a different region of the UL34 homolog, M50, in binding to the UL31 homolog, M53 (4,19,28). The interaction region is located in a highly conserved stretch of residues at the N terminus of M50 CR2. Whether the differences between MCMV M50 and HSV pUL34 reflect a very different structural basis for interaction is not yet clear.At the NE, pUL34 and pUL31 mediate subsequent steps ...