Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication

Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50. Here, we present an NMR-determined solution-state structure of the murine CMV homolog of UL50 (M50; residues 1–168) with a strikingly intricate protein fold that is matched by no other known protein folds in its entirety. Using NMR methods, we mapped the interaction of M50 with a highly conserved UL53-derived peptide, corresponding to a segment that is required for heterodimerization. The UL53 peptide binding site mapped onto an M50 surface groove, which harbors a large cavity. Point mutations of UL50 residues corresponding to surface residues in the characterized M50 heterodimerization interface substantially decreased UL50–UL53 binding in vitro, eliminated UL50–UL53 colocalization, prevented disruption of nuclear lamina, and halted productive virus replication in HCMV-infected cells. Our results provide detailed structural information on a key protein–protein interaction involved in nuclear egress and suggest that NEC subunit interactions can be an attractive drug target. Human cytomegalovirus (HCMV) is an important human pathogen. Current anti-HCMV therapies suffer from toxicities, drug resistance, and/or pharmacokinetic limitations. A possible antiviral drug target is a two-subunit complex that orchestrates nuclear egress, an essential, unusual mechanism by which nucleocapsids move from the nucleus to the cytoplasm during viral replication. We solved the structure of the conserved core of one subunit of the complex, mapped the primary interaction interface with the other subunit, and tested the importance of specific residues for subunit interactions and viral replication. The combined biophysical and biological analyses presented here develop molecular understanding of nuclear egress and identify a groove that includes a large cavity on the subunit as an attractive target for yet to be identified inhibitors.