title: Biochemical, biophysical, and mutational analyses of subunit interactions of the human cytomegalovirus nuclear egress complex.
abstract: Nuclear egress, the trafficking of herpesvirus nucleocapsids from the nucleus to the cytoplasm, involves two conserved viral proteins that form a complex at the nuclear envelope, referred to as the nuclear egress complex. In human cytomegalovirus, these two proteins are called UL50 and UL53. To study UL50 and UL53 in molecular detail, these proteins were expressed in bacteria and purified. To obtain highly expressed, pure proteins, it was necessary to truncate both constructs based on sequence conservation and predicted secondary structural elements. Size exclusion chromatography and analytical ultracentrifugation studies indicated that the truncated form of UL50 is a monomer in solution, that the truncated form of UL53 is a homodimer, and that, when mixed, the two proteins form a heterodimer. To identify residues of UL53 crucial for homodimerization and for heterodimerization with UL50, we constructed and expressed mutant forms of UL53 containing alanine substitutions in a predicted helix. Isothermal titration calorimetry was used to measure the binding affinities of the UL53 mutants to UL50. UL53 residues, the replacement of which reduced binding to UL50, form a surface on one face of the predicted helix. Moreover, most of the substitutions that reduce UL53-UL50 interactions also reduced homodimerization. Substitutions that reduced the interaction between UL50 and UL53 in vitro also reduced colocalization of full-length UL50 and UL53 at the nuclear rim in transfected cells. These results demonstrate direct protein-protein interactions between these proteins that are likely to be mediated by a helix, and they have implications for understanding nuclear egress and for drug discovery.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: My D
authors.initials: MD
authors.lastname: Sam
authors.forename: Brady T
authors.initials: BT
authors.lastname: Evans
authors.forename: Donald M
authors.initials: DM
authors.lastname: Coen
authors.forename: James M
authors.initials: JM
authors.lastname: Hogle
mesh_terms: D021581:Active Transport, Cell Nucleus; D019943:Amino Acid Substitution; D002151:Calorimetry; D003587:Cytomegalovirus / Q000502:physiology*; D019281:Dimerization; D006801:Humans; D046911:Macromolecular Substances / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D025941:Protein Interaction Mapping; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism; D014779:Virus Replication
chemical_list: D046911:Macromolecular Substances; D014764:Viral Proteins