SUMMARY Herpesvirus assembly is critically dependent on specific interactions between viral proteins. In nuclear egress, the NEC heterodimer, consisting of pUL31 and pUL34, performs multiple functions and interacts with multiple other viral factors. In many cases, however, the mechanistic consequence and significance of those interactions is unknown. Similarly, cytoplasmic envelopment depends upon interactions made by a few essential tegument proteins (VP16, pUL36 and pUL37 in HSV-1), but which of their interactions (other than with each other) are important for this process is unknown despite a wide array of interaction partners identified by proteomic and other approaches. Here we propose to use a novel directed evolution approach for functional identification of crucial interactions. We replace an essential HSV gene with its homolog from VZV and then perform a growth selection for viruses that can use the VZV homolog for assembly. Mapping of the mutations that have occurred during this directed evolution can identify novel functional interactions and establish the significance of others. We present preliminary data showing the utility of this approach using a chimeric virus in which HSV-1 UL34 is replaced by VZV ORF24. We have identified a novel functional interaction with HSV-1 ICP4 and validated the significance of a previously reported interaction with ICP22. We propose to determine the mechanism of action of these critical interactions and use the system to identify others. In addition, we propose to expand the use of this system another gene target relevant to nuclear egress, pUL31.