Identification of crucial hydrogen-bonding residues for the interaction ofherpes simplex virus DNA polymerase subunits via peptide display, mutational, and calorimetric approaches

The catalytic subunit, Pol, of herpes simplex virus DNA polymerase interact s via its extreme C terminus with the processivity subunit, UL42. This inte raction is critical for viral replication and thus a potential target for a ntiviral drug action. To investigate the Pol-binding region on UL42, we eng ineered UL42 mutations but also used random peptide display to identify art ificial ligands of the Pol C terminus. The latter approach selected ligands with homology to residues 171 to 176 of UL42, Substitution of glutamine 17 1 with alanine greatly impaired binding to Pol and stimulation of long-chai n DNA synthesis by Pol, identifying this residue as crucial for subunit int eractions. To study these interactions quantitatively, we used isothermal t itration calorimetry and wild-type and mutant forms of Pol-derived peptides and UL42. Each of three peptides corresponding to either the Last 36, 27, or 18 residues of Pol bound specifically to UL42 in a 1:1 complex with a di ssociation constant of 1 to 2 muM. Thus, the last 18 residues suffice for m ost of the binding energy, which was due mainly to a change in enthalpy. Su bstitutions at positions corresponding to Pol residue 1228 or 1229 or at UL 42 residue 171 abolished or greatly reduced binding. These residues partici pate in hydrogen bonds observed in the crystal structure of the C terminus of Pol bound to UL42. Thus, interruption of these few bonds is sufficient t o disrupt the interaction, suggesting that small molecules targeting the re levant side chains could interfere with Pol-UL42 binding.