Sensitivity of the C-Terminal Nuclease Domain of Kaposi's Sarcoma-Associated Herpesvirus ORF29 to Two Classes of Active-Site Ligands

Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma, belongs to the family, whose members employ a multicomponent terminase to resolve nonparametric viral DNA into genome-length units prior to their packaging. Homology modeling of the ORF29 C-terminal nuclease domain (pORF29C) and bacteriophage Sf6 gp2 have suggested an active site clustered with four acidic residues, D , E , D , and D , that collectively sequester the catalytic divalent metal (Mn ) and also provided important insight into a potential inhibitor binding mode. Using this model, we have expressed, purified, and characterized the wild-type pORF29C and variants with substitutions at the proposed active-site residues. Differential scanning calorimetry demonstrated divalent metal-induced stabilization of wild-type (WT) and D A pORF29C, consistent with which these two enzymes exhibited Mn -dependent nuclease activity, although the latter mutant was significantly impaired. Thermal stability of WT and D A pORF29C was also enhanced by binding of an α-hydroxytropolone (α-HT) inhibitor shown to replace divalent metal at the active site. For the remaining mutants, thermal stability was unaffected by divalent metal or α-HT binding, supporting their role in catalysis. pORF29C nuclease activity was also inhibited by two classes of small molecules reported to inhibit HIV RNase H and integrase, both of which belong to the superfamily of nucleotidyltransferases. Finally, α-HT inhibition of KSHV replication suggests ORF29 nuclease function as an antiviral target that could be combined with latency-activating compounds as a shock-and-kill antiviral strategy.