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Journal of Virology, June 2001, p. 4990-4998, Vol. 75, No. 11
Department of Biological Chemistry and
Molecular Pharmacology and Committee on Virology, Harvard Medical
School, Boston, Massachusetts 02115
Received 3 November 2000/Accepted 10 January 2001
The catalytic subunit, Pol, of herpes simplex virus DNA polymerase
interacts via its extreme C terminus with the processivity subunit,
UL42. This interaction is critical for viral replication and thus a
potential target for antiviral drug action. To investigate the
Pol-binding region on UL42, we engineered UL42 mutations
but also used random peptide display to identify artificial ligands of
the Pol C terminus. The latter approach selected ligands with homology
to residues 171 to 176 of UL42. Substitution of glutamine 171 with
alanine greatly impaired binding to Pol and stimulation of long-chain
DNA synthesis by Pol, identifying this residue as crucial for subunit
interactions. To study these interactions quantitatively, we used
isothermal titration 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 dissociation constant of 1 to 2 µM.
Thus, the last 18 residues suffice for most of the binding energy,
which was due mainly to a change in enthalpy. Substitutions at
positions corresponding to Pol residue 1228 or 1229 or at UL42 residue
171 abolished or greatly reduced binding. These residues participate 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
to disrupt the interaction, suggesting that small molecules targeting
the relevant side chains could interfere with Pol-UL42 binding.
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.11.4990-4998.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Identification of Crucial Hydrogen-Bonding Residues for the
Interaction of Herpes Simplex Virus DNA Polymerase Subunits via
Peptide Display, Mutational, and Calorimetric Approaches

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Corresponding author. Mailing address: Dept. of
Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, 250 Longwood Ave., Boston, MA 02115. Phone: (617) 432-1691. Fax: (617) 432-3833. E-mail: Don_Coen{at}hms.harvard.edu
Present address: Wyeth Research, Department of Biological
Chemistry, Cambridge, MA 02140.
Present address: Department of Immunology and Infectious Diseases,
Harvard School of Public Health, Boston, MA 02115.
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