This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Loregian, A.
Right arrow Articles by Coen, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Loregian, A.
Right arrow Articles by Coen, D. M.

 Previous Article  |  Next Article 

Journal of Virology, September 2004, p. 9084-9092, Vol. 78, No. 17
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.17.9084-9092.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Specific Residues in the Connector Loop of the Human Cytomegalovirus DNA Polymerase Accessory Protein UL44 Are Crucial for Interaction with the UL54 Catalytic Subunit

Arianna Loregian,1,2 Brent A. Appleton,1 James M. Hogle,1 and Donald M. Coen1*

Department of Biological Chemistry and Molecular Pharmacology and Committee on Virology, Harvard Medical School, Boston, Massachusetts,1 Department of Histology, Microbiology and Medical Biotechnologies, University of Padua, Padua, Italy2

Received 28 January 2004/ Accepted 26 April 2004

The human cytomegalovirus DNA polymerase includes an accessory protein, UL44, which has been proposed to act as a processivity factor for the catalytic subunit, UL54. How UL44 interacts with UL54 has not yet been elucidated. The crystal structure of UL44 revealed the presence of a connector loop analogous to that of the processivity subunit of herpes simplex virus DNA polymerase, UL42, which is crucial for interaction with its cognate catalytic subunit, UL30. To investigate the role of the UL44 connector loop, we replaced each of its amino acids (amino acids 129 to 140) with alanine. We then tested the effect of each substitution on the UL44-UL54 interaction by glutathione S-transferase pulldown and isothermal titration calorimetry assays, on the stimulation of UL54-mediated long-chain DNA synthesis by UL44, and on the binding of UL44 to DNA-cellulose columns. Substitutions that affected residues 133 to 136 of the connector loop measurably impaired the UL44-UL54 interaction without altering the ability of UL44 to bind DNA. One substitution, I135A, completely disrupted the binding of UL44 to UL54 and inhibited the ability of UL44 to stimulate long-chain DNA synthesis by UL54. Thus, similar to the herpes simplex virus UL30-UL42 interaction, a residue of the connector loop of the accessory subunit is crucial for UL54-UL44 interaction. However, while alteration of a polar residue of the UL42 connector loop only partially reduced binding to UL30, substitution of a hydrophobic residue of UL44 completely disrupted the UL54-UL44 interaction. This information may aid the discovery of small-molecule inhibitors of the UL44-UL54 interaction.

* Corresponding author. Mailing address: Department 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.

Journal of Virology, September 2004, p. 9084-9092, Vol. 78, No. 17
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.17.9084-9092.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Baltz, J. L., Filman, D. J., Ciustea, M., Silverman, J. E. Y., Lautenschlager, C. L., Coen, D. M., Ricciardi, R. P., Hogle, J. M. (2009). The Crystal Structure of PF-8, the DNA Polymerase Accessory Subunit from Kaposi's Sarcoma-Associated Herpesvirus. J. Virol. 83: 12215-12228 [Abstract] [Full Text]  
  • Alvisi, G., Roth, D. M., Camozzi, D., Pari, G. S., Loregian, A., Ripalti, A., Jans, D. A. (2009). The Flexible Loop of the Human Cytomegalovirus DNA Polymerase Processivity Factor ppUL44 Is Required for Efficient DNA Binding and Replication in Cells. J. Virol. 83: 9567-9576 [Abstract] [Full Text]  
  • Strang, B. L., Sinigalia, E., Silva, L. A., Coen, D. M., Loregian, A. (2009). Analysis of the Association of the Human Cytomegalovirus DNA Polymerase Subunit UL44 with the Viral DNA Replication Factor UL84. J. Virol. 83: 7581-7589 [Abstract] [Full Text]  
  • Sinigalia, E., Alvisi, G., Mercorelli, B., Coen, D. M., Pari, G. S., Jans, D. A., Ripalti, A., Palu, G., Loregian, A. (2008). Role of Homodimerization of Human Cytomegalovirus DNA Polymerase Accessory Protein UL44 in Origin-Dependent DNA Replication in Cells. J. Virol. 82: 12574-12579 [Abstract] [Full Text]  
  • Komazin-Meredith, G., Santos, W. L., Filman, D. J., Hogle, J. M., Verdine, G. L., Coen, D. M. (2008). The Positively Charged Surface of Herpes Simplex Virus UL42 Mediates DNA Binding. J. Biol. Chem. 283: 6154-6161 [Abstract] [Full Text]  
  • Loregian, A., Sinigalia, E., Mercorelli, B., Palu, G., Coen, D. M. (2007). Binding parameters and thermodynamics of the interaction of the human cytomegalovirus DNA polymerase accessory protein, UL44, with DNA: implications for the processivity mechanism. Nucleic Acids Res 35: 4779-4791 [Abstract] [Full Text]  
  • Appleton, B. A., Brooks, J., Loregian, A., Filman, D. J., Coen, D. M., Hogle, J. M. (2006). Crystal Structure of the Cytomegalovirus DNA Polymerase Subunit UL44 in Complex with the C Terminus from the Catalytic Subunit: DIFFERENCES IN STRUCTURE AND FUNCTION RELATIVE TO UNLIGANDED UL44. J. Biol. Chem. 281: 5224-5232 [Abstract] [Full Text]  
  • Tchesnokov, E. P., Gilbert, C., Boivin, G., Gotte, M. (2006). Role of Helix P of the Human Cytomegalovirus DNA Polymerase in Resistance and Hypersusceptibility to the Antiviral Drug Foscarnet. J. Virol. 80: 1440-1450 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»