title: Characterization of the C-Terminal Nuclease Domain of Herpes Simplex Virus pUL15 as a Target of Nucleotidyltransferase Inhibitors.
abstract: The natural product α-hydroxytropolones manicol and β-thujaplicinol inhibit replication of herpes simplex viruses 1 and 2 (HSV-1 and HSV-2, respectively) at nontoxic concentrations. Because these were originally developed as divalent metal-sequestering inhibitors of the ribonuclease H activity of HIV-1 reverse transcriptase, α-hydroxytropolones likely target related HSV proteins of the nucleotidyltransferase (NTase) superfamily, which share an "RNase H-like" fold. One potential candidate is pUL15, a component of the viral terminase molecular motor complex, whose C-terminal nuclease domain, pUL15C, has recently been crystallized. Crystallography also provided a working model for DNA occupancy of the nuclease active site, suggesting potential protein-nucleic acid contacts over a region of ∼ 14 bp. In this work, we extend crystallographic analysis by examining pUL15C-mediated hydrolysis of short, closely related DNA duplexes. In addition to defining a minimal substrate length, this strategy facilitated construction of a dual-probe fluorescence assay for rapid kinetic analysis of wild-type and mutant nucleases. On the basis of its proposed role in binding the phosphate backbone, studies with pUL15C variant Lys700Ala showed that this mutation affected neither binding of duplex DNA nor binding of small molecule to the active site but caused a 17-fold reduction in the turnover rate (kcat), possibly by slowing conversion of the enzyme-substrate complex to the enzyme-product complex and/or inhibiting dissociation from the hydrolysis product. Finally, with a view of pUL15-associated nuclease activity as an antiviral target, the dual-probe fluorescence assay, in combination with differential scanning fluorimetry, was used to demonstrate inhibition by several classes of small molecules that target divalent metal at the active site.
authors.affiliation: Basic Research Laboratory, National Cancer Institute , Frederick, Maryland 21702, United States.
authors.forename: Takashi
authors.initials: T
authors.lastname: Masaoka
authors.affiliation: Department of Molecular Biosciences, University of Kansas , Lawrence, Kansas 66045, United States.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Department of Chemistry, Brooklyn College, City University of New York , Brooklyn, New York 11210, United States.|Ph.D. Program in Chemistry, The Graduate Center of the City University of New York , New York, New York 10016, United States.
authors.forename: Danielle R
authors.initials: DR
authors.lastname: Hirsch
authors.affiliation: Department of Chemistry, Brooklyn College, City University of New York , Brooklyn, New York 11210, United States.|Ph.D. Program in Chemistry, The Graduate Center of the City University of New York , New York, New York 10016, United States.
authors.forename: Michael P
authors.initials: MP
authors.lastname: D'Erasmo
authors.affiliation: Department of Chemistry, Brooklyn College, City University of New York , Brooklyn, New York 11210, United States.|Ph.D. Program in Chemistry, The Graduate Center of the City University of New York , New York, New York 10016, United States.
authors.forename: Christine
authors.initials: C
authors.lastname: Meck
authors.affiliation: Department of Molecular Biosciences, University of Kansas , Lawrence, Kansas 66045, United States.
authors.forename: Brittany
authors.initials: B
authors.lastname: Varnado
authors.affiliation: Department of Molecular Microbiology and Immunology, St. Louis University School of Medicine , St. Louis, Missouri 63104, United States.
authors.forename: Ankit
authors.initials: A
authors.lastname: Gupta
authors.affiliation: Department of Chemistry, St. Louis University , St. Louis, Missouri 63103, United States.
authors.forename: Marvin J
authors.initials: MJ
authors.lastname: Meyers
authors.affiliation: School of Veterinary Medicine, Louisiana State University , Baton Rouge, Louisiana 70803, United States.
authors.forename: Joel
authors.initials: J
authors.lastname: Baines
authors.affiliation: Molecular Targets Laboratory, National Cancer Institute , Frederick, Maryland 21702, United States.
authors.forename: John A
authors.initials: JA
authors.lastname: Beutler
authors.affiliation: Department of Chemistry, Brooklyn College, City University of New York , Brooklyn, New York 11210, United States.|Ph.D. Program in Chemistry, The Graduate Center of the City University of New York , New York, New York 10016, United States.
authors.forename: Ryan P
authors.initials: RP
authors.lastname: Murelli
authors.affiliation: Department of Molecular Biosciences, University of Kansas , Lawrence, Kansas 66045, United States.
authors.forename: Liang
authors.initials: L
authors.lastname: Tang
authors.affiliation: Basic Research Laboratory, National Cancer Institute , Frederick, Maryland 21702, United States.
authors.forename: Stuart F J
authors.initials: SF
authors.lastname: Le Grice
mesh_terms: D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005453:Fluorescence; D018259:Herpesvirus 1, Human / Q000737:chemistry*; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors*; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D004791:Enzyme Inhibitors; D014764:Viral Proteins; C069185:DNA cleavage and packaging proteins, Herpesvirus; D009713:Nucleotidyltransferases
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