The long-term objective of this research is a detailed understanding of herpes virus DNA polymerases and drugs that target them. These enzymes, which include a catalytic subunit (Pol) and an accessory subunit that stimulates long-chain DNA synthesis, are both prototype ?.-like DNA polymerases and excellent targets for antiviral drugs. This latter property is especially health-related, as new drugs are needed for treatment of herpes virus infections. In this application, unanswered questions regarding accessory subunits, catalytic subunits, and drugs that target these proteins and their interaction are addressed.
Specific aim 1 is to investigate the unusual and different manners by which the accessory subunits, herpes simplex virus (HSV) UL42 and human cytomegalovirus (HCMV) UL44, interact with DNA so that they bind tightly, yet diffuse linearly along the DNA to permit possessive DNA synthesis. Single-molecule approaches will be used to analyze how these proteins move on DNA, particularly whether UL44 """"""""slides"""""""" or whether it """"""""hops"""""""" like UL42, and whether these proteins necessarily move helically or can, for example, move along one side of the helix. The force required to move these subunits will be assessed. Studies will be initiated to measure the force required to stop or slow the catalytic subunits, and to optimize crystallization conditions of protein-DNA complexes in preparation for structure determination.
Specific aim 2 is to investigate the roles of structural domains of the catalytic subunits that are N-terminal to the thumb, palm, and fingers domains in terms of enzymatic functions, viral replication, and mechanisms of antiviral drug resistance. Two structural domains, pre-NH2 and NH2, have been observed in the crystal structure of HSV Pol, but their roles in enzyme function and viral replication are unknown. Additionally, the mechanisms by which mutant HCMV Pols with substitutions in their 3'-5'exonuclease domain resist ganciclovir (GCV) action are not understood. To address these questions, mutant enzymes will be engineered and assayed for relevant enzyme activities. Interesting HSV pol mutations will be engineered into mutant viruses in preparation for assays of their effects on viral replication.
Specific aim 3 is to study the mechanisms of promising new compounds that inhibit the interaction of HCMV Pol and UL44 and HCMV replication. A combination of genetic, virological, biochemical, and crystallographic approaches will be initiated. Efforts to discover new agents that exploit this interaction (and to understand their mechanisms) will also begin.

Public Health Relevance

Herpes simplex virus (HSV) causes widespread disease in the population at large, and both HSV and human cytomegalovirus (HCMV) cause severe disease in people with impaired immunity. There is considerable need for new drugs to combat HCMV and drug-resistant HSV. The research proposed not only should provide information that could aid in better understanding of these pathogens and in understanding mechanisms of action and resistance of existing antiviral drugs, but aims directly to discover new anti- HCMV drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI019838-25
Application #
7842610
Study Section
Virology - A Study Section (VIRA)
Program Officer
Beisel, Christopher E
Project Start
1983-04-01
Project End
2011-04-30
Budget Start
2010-05-01
Budget End
2011-04-30
Support Year
25
Fiscal Year
2010
Total Cost
$423,750
Indirect Cost
Name
Harvard University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
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
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
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
Terrell, Shariya L; Pesola, Jean M; Coen, Donald M (2014) Roles of conserved residues within the pre-NH2-terminal domain of herpes simplex virus 1 DNA polymerase in replication and latency in mice. J Gen Virol 95:940-7
Strang, Blair L; Boulant, Steeve; Chang, Lynne et al. (2012) Human cytomegalovirus UL44 concentrates at the periphery of replication compartments, the site of viral DNA synthesis. J Virol 86:2089-95
Terrell, Shariya L; Coen, Donald M (2012) The pre-NH(2)-terminal domain of the herpes simplex virus 1 DNA polymerase catalytic subunit is required for efficient viral replication. J Virol 86:11057-65

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