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Journal of Virology, August 2009, p. 7581-7589, Vol. 83, No. 15
0022-538X/09/$08.00+0     doi:10.1128/JVI.00663-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Analysis of the Association of the Human Cytomegalovirus DNA Polymerase Subunit UL44 with the Viral DNA Replication Factor UL84{triangledown}

Blair L. Strang,1 Elisa Sinigalia,2 Laurie A. Silva,1 Donald M. Coen,1* and Arianna Loregian2*

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115,1 Department of Histology, Microbiology and Medical Biotechnologies, University of Padua, 35121 Padua, Italy2

Received 31 March 2009/ Accepted 14 May 2009

The central enzyme responsible for human cytomegalovirus (HCMV) DNA synthesis is a virally encoded DNA polymerase that includes a catalytic subunit, UL54, and a homodimeric accessory subunit, UL44, the presumptive HCMV DNA polymerase processivity factor. The structure of UL44 is similar to that of the eukaryotic processivity factor proliferating cell nuclear antigen (PCNA), which interacts with numerous other proteins required for faithful DNA replication. We sought to determine whether, like PCNA, UL44 is capable of interacting with multiple DNA replication proteins and, if so, whether these proteins bind UL44 at the site corresponding to where multiple proteins bind to PCNA. Initially, several proteins, including the viral DNA replication factors UL84 and UL57, were identified by mass spectrometry in immunoprecipitates of UL44 from infected cell lysate. The association of UL44/UL84, but not UL44/UL57, was confirmed by reciprocal coimmunoprecipitation of these proteins from infected cell lysates and was resistant to nuclease treatment. Yeast two-hybrid analyses demonstrated that the substitution of residues in UL44 that prevent UL44 homodimerization or abrogate the binding of UL54 to UL44 do not abrogate the UL44/UL84 interaction. Reciprocal glutathione-S-transferase (GST) pulldown experiments using bacterially expressed UL44 and UL84 confirmed these results and, further, demonstrated that a UL54-derived peptide that competes with UL54 for UL44 binding does not prevent the association of UL84 with UL44. Taken together, our results strongly suggest that UL44 and UL84 interact directly using a region of UL44 different from the UL54 binding site. Thus, UL44 can bind interacting replication proteins using a mechanism different from that of PCNA.

* Corresponding author. Mailing address for Arianna Loregian: Department of Histology, Microbiology and Medical Biotechnologies, University of Padua, 35121 Padua, Italy. Phone: 39 049 8272363. Fax: 39 049 8272355. E-mail: arianna.loregian{at}unipd.it. Mailing address for Donald M. Coen: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115. Phone: (617) 432-1691. Fax: (617) 432-3833. E-mail: don_coen{at}hms.harvard.edu

{triangledown} Published ahead of print on 20 May 2009.

Journal of Virology, August 2009, p. 7581-7589, Vol. 83, No. 15
0022-538X/09/$08.00+0     doi:10.1128/JVI.00663-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Kaiser, N., Lischka, P., Wagenknecht, N., Stamminger, T. (2009). Inhibition of Human Cytomegalovirus Replication via Peptide Aptamers Directed against the Nonconventional Nuclear Localization Signal of the Essential Viral Replication Factor pUL84. J. Virol. 83: 11902-11913 [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]  


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