Sensitivity of the C-terminal nuclease domain of Kaposi's sarcoma-associated herpesvirus ORF29 to two classes of active site ligands [PublishAheadOfPrint]

Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma, belongs to the Herpesviridae family, whose members employ a multicomponent terminase to resolve nonparametric viral DNA into genome-length units prior to their packaging. Homology modeling of ORF29 C-terminal nuclease domain (pORF29C) and bacteriophage Sf6 gp2 have suggested an active site clustered with four acidic residues, D⁴⁷⁶, E⁵⁵⁰, D⁶⁶¹ and D⁶⁶² 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 substituted at the proposed active site residues. Differential scanning calorimetry demonstrated divalent metal-induced stabilization of WT and D⁶⁶¹A pORF29C, consistent with which these two enzymes exhibited Mn⁺⁺-dependent nuclease activity, although the latter mutant was significantly impaired. Thermal stability of WT and D⁶⁶¹A 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 ribonuclease H and integrase, both of which belong to the superfamily of nucleotidyltransferases. Finally, α-HT inhibition of KSHV replication suggests ORF29 nuclease function as a potential antiviral target that could be combined with latency-activating compounds as a "shock-and-kill" antiviral strategy.

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