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    Characterization of the C-Terminal Nuclease Domain of Herpes Simplex Virus pUL15 as a Target of Nucleotidyltransferase Inhibitors

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    Issue Date
    2016-02-09
    Author
    Masaoka, Takashi
    Zhao, Haiyan
    Hirsch, Danielle R.
    D'Erasmo, Michael P.
    Meck, Christine
    Varnado, Brittany
    Gupta, Ankit
    Meyers, Marvin J.
    Baines, Joel D.
    Beutler, John A.
    Murelli, Ryan P.
    Tang, Liang
    Le Grice, Stuart F. J.
    Publisher
    ACS
    Type
    Article
    Article Version
    Scholarly/refereed, author accepted manuscript
    Rights
    Copyright © 2016 American Chemical Society
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    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.
    URI
    http://hdl.handle.net/1808/23749
    DOI
    https://doi.org/10.1021/acs.biochem.5b01254
    Collections
    • Molecular Biosciences Scholarly Works [537]
    Citation
    Masaoka, T., Zhao, H., Hirsch, D. R., D’Erasmo, M. P., Meck, C., Varnado, B., … Le Grice, S. F. J. (2016). Characterization of the C-Terminal Nuclease Domain of Herpes Simplex Virus pUL15 as a Target of Nucleotidyltransferase Inhibitors. Biochemistry, 55(5), 809–819. http://doi.org/10.1021/acs.biochem.5b01254

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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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