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.

Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage

“…These results support a model whereby certain compounds with chelating motifs can interact with the divalent cations in the pUL89-C active site to inhibit endonuclease activity. However, inhibition of HSV-1 pUL15C endonuclease activity by ␤-thujaplicinol and related compounds featuring a chelating motif was equivalent regardless of the order of the addition of reagents or preincubation when examined by using a dual-probe fluorescence biochemical assay (19). Differences in compounds, assays, and enzymes can account for these results.…”

“…How pUL89 recognizes dsDNA in the presence or the absence of pUL56 and other viral proteins remains unknown and warrants further investigation, since our current inhibitor, compound 10k, may be competitive with a substrate. The pUL89-C homolog HSV-1 pUL15C was found to cleave a double-strand DNA substrate as small as 14 to 15 bp (19). The substrates used contained sequence elements designed to mimic aspects of the viral pac sites, sites that contribute to viral genomic cleavage and packaging, whereas our substrate did not.…”

“…The pUL89-C nuclease function was blocked by the human immunodeficiency virus (HIV) IN inhibitor raltegravir (15), suggesting that inhibiting pUL89-C may be possible by using a similar metal-chelating pharmacophore. Recently, compounds with metal-chelating motifs were identified as inhibitors of herpesviruses (16)(17)(18) and in a separate study were identified as inhibitors of HSV-1 pUL15C (19). However, the likely target(s) of the inhibitors in infected cells has not been identified.…”

“…Compounds containing chelating motifs could bind any of several viral or cellular metal-binding proteins or even proteins that may not be metal binding in cells. An example is raltegravir, a weak inhibitor of pUL89-C (15) but a potent pUL15C inhibitor in biochemical assays (19). HSV-1 resistance to raltegravir maps to a viral DNA polymerase accessory factor and not UL15 (20), indicating that raltegravir likely binds the accessory factor in the infected cell.…”

Broad anti-herpesviral activity of α-hydroxytropolones

“…While tropolones are well known metalloenzyme inhibitors due to the high negative charge character on both the carbonyl and adjacent oxygen at physiological pH, αHTs provide an additional contiguous oxygen that makes them particularly good inhibitors of dinuclear metalloenzymes (Bentley, 2008; Piettre et al, 1997). To investigate the nature of αHT HSV-1 inhibitory activity, one specific HSV-1 NTS-like enzyme, pU L 15C of the viral terminase, was assessed in the presence of several synthetic β-thujaplicinol derivatives through the analysis of pU L 15C-mediated hydrolysis of short DNA duplexes using dual-probe fluorescence (Masaoka et al, 2016). Interestingly, some of the most potent pU L 15C synthetic αHTs inhibited HSV-1 replication only modestly, while those that strongly inhibited HSV-1 replication had poor anti-terminase activity (Ireland et al, 2016; Masaoka et al, 2016).…”