The long-term objective of this research is a detailed understanding of herpesvirus DNA polymerases. These enzymes, which include a catalytic subunit (Pol) and an accessory subunit that stimulates long-chain DNA synthesis, are prototype alpha-like DNA polymerases and excellent targets for antiviral drugs. New drugs are needed for treatment of herpesvirus infections in patients with AIDS, especially those caused by human cytomegalovirus (CMV).
Specific aim 1 is to determine mechanisms that regulate translation of HSV Pol and their importance to the virus. Mutational, RNA-binding, and translational analyses will test the hypotheses that a virion protein, US11, stimulates Pol translation early in infection, while inefficient translation later is beneficial to the virus.
Specific aim 2 is to analyze functions of the small C-terminal domain of HSV Pol and to determine the mechanism by which the accessory subunit, UL42, enhances processivity despite stable UL42-DNA binding. Effects of mutations on DNA-binding by the C-terminal domain will be correlated with effects on Pol activity and virus replication. UL42 mechanisms will be examined using limited proteolysis and DNA footprinting.
Specific aim 3 is to determine mechanisms by which mutant CMV Pols resist ganciclovir (GCV) action, by analyzing Pol interactions with GCV-TP and GCV-containing DNA.
Specific aim 4 is to investigate CMV Pol's interaction with its accessory protein, UL44. Interacting residues will be defined genetically and tested for their importance in CMV DNA replication. This information will serve as a starting point for discovery of antiviral drugs.
Specific aim 5 is to solve the three dimensional structures of domains of HSV Pol and UL42 and CMV Pol and UL44 using x-ray crystallography to gain basic information regarding polymerase functions and as a starting point for drug discovery.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI019838-16
Application #
2886431
Study Section
Virology Study Section (VR)
Program Officer
Beisel, Christopher E
Project Start
1983-04-01
Project End
2002-03-31
Budget Start
1999-04-01
Budget End
2000-03-31
Support Year
16
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Harvard University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Lawler, Jessica L; Mukherjee, Purba; Coen, Donald M (2018) Herpes Simplex Virus 1 DNA Polymerase RNase H Activity Acts in a 3'-to-5' Direction and Is Dependent on the 3'-to-5' Exonuclease Active Site. J Virol 92:
Beelontally, Rooksarr; Wilkie, Gavin S; Lau, Betty et al. (2017) Identification of compounds with anti-human cytomegalovirus activity that inhibit production of IE2 proteins. Antiviral Res 138:61-67
Strang, Blair L (2017) RO0504985 is an inhibitor of CMGC kinase proteins and has anti-human cytomegalovirus activity. Antiviral Res 144:21-26
Chen, Han; Coseno, Molly; Ficarro, Scott B et al. (2017) A Small Covalent Allosteric Inhibitor of Human Cytomegalovirus DNA Polymerase Subunit Interactions. ACS Infect Dis 3:112-118
Khan, Amina S; Murray, Matthew J; Ho, Catherine M K et al. (2017) High-throughput screening of a GlaxoSmithKline protein kinase inhibitor set identifies an inhibitor of human cytomegalovirus replication that prevents CREB and histone H3 post-translational modification. J Gen Virol 98:754-768
Wilkie, Adrian R; Lawler, Jessica L; Coen, Donald M (2016) A Role for Nuclear F-Actin Induction in Human Cytomegalovirus Nuclear Egress. MBio 7:
Chen, Han; Li, Chengwei; Zemlicka, Jiri et al. (2016) Potency and Stereoselectivity of Cyclopropavir Triphosphate Action on Human Cytomegalovirus DNA Polymerase. Antimicrob Agents Chemother 60:4176-82
Polachek, William S; Moshrif, Hanan F; Franti, Michael et al. (2016) High-Throughput Small Interfering RNA Screening Identifies Phosphatidylinositol 3-Kinase Class II Alpha as Important for Production of Human Cytomegalovirus Virions. J Virol 90:8360-71
Bender, Brian J; Coen, Donald M; Strang, Blair L (2014) Dynamic and nucleolin-dependent localization of human cytomegalovirus UL84 to the periphery of viral replication compartments and nucleoli. J Virol 88:11738-47
Chen, Han; Beardsley, G Peter; Coen, Donald M (2014) Mechanism of ganciclovir-induced chain termination revealed by resistant viral polymerase mutants with reduced exonuclease activity. Proc Natl Acad Sci U S A 111:17462-7

Showing the most recent 10 out of 87 publications