|Keywords:||Biology, Virology; Biology, Molecular|
|Full text PDF:||http://nrs.harvard.edu/urn-3:HUL.InstRepos:14226065|
The Herpesviridae family of viruses includes a number of human pathogens of clinical importance. Like other herpesviruses, cytomegaloviruses require a heterodimeric nuclear egress complex (NEC) consisting of a membrane-bound protein and a soluble nucleoplasmic protein, termed in murine cytomegalovirus (MCMV) M50 and M53, respectively. Genetic, electron microscopic, and immunocytochemical studies have revealed the importance of this complex for viral replication, most predominantly in facilitating egress of viral nucleocapsids across the nuclear membrane. Despite the significance of the NEC to the herpesvirus life cycle, there is a dearth of structural information regarding the components of the complex. We present here an NMR-determined solution-state structure of the conserved, structured, soluble portion of M50 (residues 1-168), which exhibits novel structural character. We mapped the binding site of a highly conserved minimal binding domain of the M53 homologue from human cytomegalovirus (HCMV; UL53) required for heterodimerization onto the structure and identified specific residues in a groove within the M50 protein fold that interact with the UL53 peptide. This site was verified biophysically and biologically: single amino acid substitutions of the corresponding residues of the homologous protein from HCMV (UL50) resulted in decreased UL53 binding in vitro, as measured by isothermal titration calorimetry, and substitutions that had the greatest effect on binding affinity caused disruption of UL50-UL53 co-localization and lethal defects in the context of HCMV infection. We then compared the effect of binding UL53 peptide with binding of the larger natural binding partner, M53 (residues 103-333) via NMR, with the results suggesting that conformational changes most likely occur on a fold-wide level in the context of the full complex. We suggest that these findings combined with the clinical relevance, the virus-specific aspects of nuclear egress, and the novelty of the structure make the HCMV NEC an attractive potential drug target. To this end, we used in silico screening to identify possible small molecule inhibitors and have begun validating top screen hits biophysically and biologically.