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Disruption of the interaction between the HCMV DNA polymerase subunits: towards new anti-HCMV inhibitors

Abstract number: 902_p552

Loregian A.

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Objectives:

The human cytomegalovirus DNA polymerase is composed of a catalytic subunit, UL54, and an accessory protein, UL44. The observations that both UL54 and UL44 are essential for HCMV DNA replication, and that antisense inhibition of UL44 synthesis in HCMV-infected cells strongly inhibits viral DNA replication raises the possibility that the UL54/UL44 interaction might be a valid target for antiviral drugs.

Methods and Results

To investigate this possibility, overlapping peptides spanning residues 1161–1242 of UL54 were synthesised and tested for inhibition of the interaction between purified UL54 and UL44 proteins. A peptide, LPRRLHLEPAFLPYSVKAHECC, corresponding to residues 1221–1242 at the very C-terminus of UL54, disrupted both the physical interaction between the two proteins and specifically inhibited the stimulation of UL54 by UL44. Moreover, to define individual residues in UL44 and UL54 that are crucial for interacting with each other, we have engineered several mutations both in the C-terminal region of UL54 and in a region of UL44 identified in the crystal structure as the ‘connector loop’. Substitution of alanine for Ile135 in UL44 or for Leu1227 or Phe1231 in UL54 greatly and specifically impaired the UL54–UL44 interaction in both pull-down assays and assays of long-chain DNA synthesis, identifying these residues as crucial for subunit interaction.

Conclusions:

Thus, a few specific side chains appear to be crucial for UL54/UL44 interaction, suggesting that small molecules targeting the relevant side chains could interfere with this interaction. This information may aid in the discovery of new drugs for the treatment of HCMV infection.

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Session Details

Date: 01/08/2007
Time: 00:00-00:00
Session name: XXIst ISTH Congress
Subject:
Location: Oxford, UK
Presentation type:
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