Residues of Human Cytomegalovirus DNA Polymerase Catalytic Subunit UL54 That Are Necessary and Sufficient for Interaction with the Accessory Protein UL44

Abstract

The human cytomegalovirus DNA polymerase contains a catalytic subunit, UL54, and an accessory protein, UL44. Recent studies suggested that UL54 might interact via its extreme C terminus with UL44 (A. Loregian, R. Rigatti, M. Murphy, E. Schievano, G. Palu', and H. S. Marsden, J. Virol. 77:8336-8344, 2003). To address this hypothesis, we quantitatively measured the binding of peptides corresponding to the extreme C terminus of UL54 to UL44 by using isothermal titration calorimetry. A peptide corresponding to the last 22 residues of UL54 was sufficient to bind specifically to UL44 in a 1:1 complex with a dissociation constant of ca. 0.7 M. To define individual residues in this segment that are crucial for interacting with UL44, we engineered a series of mutations in the C-terminal region of UL54. The UL54 mutants were tested for their ability to interact with UL44 by glutathione S-transferase pulldown assays, for basal DNA polymerase activity, and for long-chain DNA synthesis in the presence of UL44. We observed that deletion of the C-terminal segment or substitution of alanine for Leu1227 or Phe1231 in UL54 greatly impaired both the UL54-UL44 interaction in pulldown assays and long-chain DNA synthesis without affecting basal polymerase activity, identifying these residues as important for subunit interaction. Thus, like the herpes simplex virus UL30-UL42 interaction, a few specific side chains in the C terminus of UL54 are crucial for UL54-UL44 interaction. However, the UL54 residues important for interaction with UL44 are hydrophobic and not basic. This information might aid in the rational design of new drugs for the treatment of human cytomegalovirus infection.Most biological processes depend on the coordinated formation of protein-protein interactions. Aside from their importance for viral biology, several interactions between viral proteins have been proposed as attractive targets for antiviral drug discovery, since the exquisite specificity of such cognate interactions affords the possibility of interfering with them in a highly specific and effective manner (for a review, see reference 16). There is a considerable need for new anti-human cytomegalovirus (HCMV) drugs since, although there are agents, most of which target the polymerization activity of the viral DNA polymerase, their use is limited by pharmacokinetic issues, antiviral resistance, and toxicity (4). A potential novel target for anti-HCMV drugs could be the interaction between the two subunits of the viral DNA polymerase.Analogous to the DNA polymerase of other herpesviruses, the HCMV DNA polymerase is composed of a 1,242-residue catalytic subunit, Pol or UL54 (13,14), and the 433-residue accessory protein UL44 (9). The UL54 protein, which is the homolog of herpes simplex virus type 1 (HSV-1) UL30, has DNA-dependent DNA polymerase activity and 3Ј-5Ј exonuclease activity (3,18,20). The UL44 accessory subunit, which is analogous to the HSV-1 UL42 protein, has been shown to bind double-stranded DNA, to specifically interact with UL54, and to stimul...

Crystal Structure of Epstein-Barr Virus DNA Polymerase Processivity Factor BMRF1

“…The C-terminal region of the polymerase catalytic subunit is critically important for interactions between the polymerase catalytic subunit and the processivity factor in the complexes of HSV-1 UL42-UL30 (46) and HCMV UL44-UL54 (47). Although the structure of the whole complex between the polymerase catalytic subunit and the processivity factor is still unavailable, the complex structures of processivity factors with the extreme C-terminal fragments of the polymerase catalytic subunit have been solved (45,48).…”

“…Furthermore, the putative BMRF1⅐ BALF5 contact region includes at least the BMRF1 dimer interface. However, in the case of UL44-UL54, the C-terminal fragment is sufficient to form a complex (47), and its interaction region does not involve the dimer interface. This fact suggests that BMRF1 and UL44 utilize different contact surfaces with the polymerase catalytic subunit, although both can form dimers in solution.…”

Crystal Structure of the Cytomegalovirus DNA Polymerase Subunit UL44 in Complex with the C Terminus from the Catalytic Subunit

“…UL54, a member of the polymerase ␣ family, displays DNA-dependent DNA polymerase and 3Ј-5Ј exonuclease activities (8 -10). UL44 is analogous to the processivity factor UL42 (11), as it binds double-stranded DNA, specifically interacts with UL54, and stimulates long chain DNA synthesis by UL54 (7,(12)(13)(14)(15).Although not yet rigorously proven by template challenge experiments, UL44 is believed to serve as the processivity factor for the polymerase.The crystal structure of residues 1-290 of UL44 (UL44⌬C290) (15) showed that UL44 has a fold remarkably similar to that of other processivity factors, including HSV UL42 (16) and monomers of the sliding clamps such as PCNA (17, 18), even though these proteins have no obvious sequence homology. Thus, each subunit of UL44, UL42, and PCNA consists of two topologically similar domains.…”

“…Thus, each subunit of UL44, UL42, and PCNA consists of two topologically similar domains. The two domains share a central ␤-sheet and are connected by a long connector loop running lengthwise across the front face of the molecule.UL44⌬C290 displays all known biochemical activities of full-length UL44 in vitro (12,19). Both UL44 and UL42 bind directly to DNA with nanomolar affinity in a manner that does not require ATP hydrolysis or accessory proteins, and the binding interaction has no apparent sequence specificity (15, 20 -23).…”

“…UL54, a member of the polymerase ␣ family, displays DNA-dependent DNA polymerase and 3Ј-5Ј exonuclease activities (8 -10). UL44 is analogous to the processivity factor UL42 (11), as it binds double-stranded DNA, specifically interacts with UL54, and stimulates long chain DNA synthesis by UL54 (7,(12)(13)(14)(15).…”