High level expression of DNA polymerases from herpesviruses

Ertl, Peter; Thomas, Mélanie; Powell, Kenneth L.

doi:10.1099/0022-1317-72-7-1729

Cited by 30 publications

(25 citation statements)

References 21 publications

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“…For the purpose of biochemical screens, herpesviridae DNA polymerases are difficult to overexpress in heterologous expression systems and have limited solubility. Hence, it has been difficult to characterize structural and functional details of these polymerases [2,3,4,5,6,7,8]. Of the eight human herpesvirus DNA polymerases, the best-studied is perhaps UL30 from HSV1.…”

Section: Introductionmentioning

confidence: 99%

Utility of the Bacteriophage RB69 Polymerase gp43 as a Surrogate Enzyme for Herpesvirus Orthologs

Bennett

Götte

2013

Viruses

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Viral polymerases are important targets in drug discovery and development efforts. Most antiviral compounds that are currently approved for treatment of infection with members of the herpesviridae family were shown to inhibit the viral DNA polymerase. However, biochemical studies that shed light on mechanisms of drug action and resistance are hampered primarily due to technical problems associated with enzyme expression and purification. In contrast, the orthologous bacteriophage RB69 polymerase gp43 has been crystallized in various forms and therefore serves as a model system that provides a better understanding of structure–function relationships of polymerases that belong the type B family. This review aims to discuss strengths, limitations, and opportunities of the phage surrogate with emphasis placed on its utility in the discovery and development of anti-herpetic drugs.

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Section: Introductionmentioning

confidence: 99%

Utility of the Bacteriophage RB69 Polymerase gp43 as a Surrogate Enzyme for Herpesvirus Orthologs

Bennett

Götte

2013

Viruses

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“…7. Lanes 1,4,7,10,13,16,19,22,25,28, and 31 contain wild-type or mutant UL54 alone; lanes 2,5,8,11,14,17,20,23,26,29, and 32 and lanes 3,6,9,12,15,18,21,24,27,30, and 33 contain wild-type or mutant UL54 plus 400 or 800 fmol of UL44, respectively. For the interaction between the C-terminal UL54 peptide and UL44, we measured a dissociation constant of ϳ0.7 M. Similar K d (1 to 2 M) and thus ⌬G values (Ϫ7.9 to Ϫ8.4 kcal/mol) were previously obtained for the binding of C-terminal UL30 peptides to UL42 (2).…”

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confidence: 99%

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

Loregian

Appleton

Hogle

et al. 2004

J Virol

114

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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...

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“…UL54 and UL44 have been expressed both in an in vitrocoupled transcription-translation reticulocyte lysate system (11) and in insect cells infected with recombinant baculoviruses (16,17). However, these approaches, due to low expression level (11) and relatively low yields and activity of the products (16), did not provide a reliable source of enzyme suitable for detailed studies.…”

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confidence: 99%

Inhibition of Human Cytomegalovirus DNA Polymerase by C-Terminal Peptides from the UL54 Subunit

Loregian

Rigatti

Murphy³

et al. 2003

J Virol

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In common with other herpesviruses, the human cytomegalovirus (HCMV) DNA polymerase contains a catalytic subunit (Pol or UL54) and an accessory protein (UL44) that is thought to increase the processivity of the enzyme. The observation that antisense inhibition of UL44 synthesis in HCMV-infected cells strongly inhibits viral DNA replication, together with the structural similarity predicted for the herpesvirus processivity subunits, highlights the importance of the accessory protein for virus growth and raises the possibility that the UL54/UL44 interaction might be a valid target for antiviral drugs. To investigate this possibility, overlapping peptides spanning residues 1161 to 1242 of UL54 were synthesized and tested for inhibition of the interaction between purified UL54 and UL44 proteins. A peptide, LPRRLHLEPAFLPYSVKAHECC, corresponding to residues 1221 to 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. A mutant peptide lacking the two carboxy-terminal cysteines was markedly less inhibitory, suggesting a role for these residues in the UL54/UL44 interaction. Circular dichroism spectroscopy indicated that the UL54 C-terminal peptide can adopt a partially ␣-helical structure. Taken together, these results indicate that the two subunits of HCMV DNA polymerase most likely interact in a way which is analogous to that of the two subunits of herpes simplex virus DNA polymerase, even though there is no sequence homology in the binding site, and suggest that the UL54 peptide, or derivatives thereof, could form the basis for developing a new class of anti-HCMV inhibitors that act by disrupting the UL54/UL44 interaction.Human cytomegalovirus (HCMV) is a human pathogen responsible for a variety of severe diseases in immunocompromised patients, including pneumonia, gastrointestinal disease, and retinitis in transplant recipients and in patients with AIDS (7). HCMV is also a major cause of congenital defects in newborn children (51). Antiviral agents currently licensed for the treatment of HCMV infections include ganciclovir, foscarnet, and cidofovir, which all inhibit the HCMV DNA polymerase (21). Ganciclovir and cidofovir are nucleoside analogs which function as DNA chain terminators, whereas foscarnet inhibits viral DNA polymerase through binding to its pyrophosphate binding site (12). There is renewed interest in the search for new HCMV inhibitors because of the emergence of drug-resistant viral strains, particularly in immunocompromised patients (42), and because some of these antiviral agents, e.g., ganciclovir and foscarnet, have toxic side effects. Since the HCMV DNA polymerase represents a major target for antiviral chemotherapy, further studies on this enzyme could be important in developing novel drugs.HCMV DNA replication has not been as fully characterized as that of herpes simplex virus (HSV); however, HCMV homologs to six of the seven proteins essential for HSV type 1 (HSV-1) replication (53) have...

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High level expression of DNA polymerases from herpesviruses

Cited by 30 publications

References 21 publications

Utility of the Bacteriophage RB69 Polymerase gp43 as a Surrogate Enzyme for Herpesvirus Orthologs

Utility of the Bacteriophage RB69 Polymerase gp43 as a Surrogate Enzyme for Herpesvirus Orthologs

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

Inhibition of Human Cytomegalovirus DNA Polymerase by C-Terminal Peptides from the UL54 Subunit

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