| Poxviruses are large double-stranded DNA viruses of metazoans and represent a threat to human populations in the form of bioterrorism and emerging infectious diseases. The first steps of poxvirus DNA synthesis yield concatemeric arrays of covalently linked genomes. The virus-encoded Holliday junction resolving enzyme, or resolvase, is required to process concatemers into unit-length genomes for packaging and thus represents an attractive drug target. Poxvirus resolvase is a member of the RNase H-like superfamily of nucleotidyl phosphotransfer enzymes, which include RNase H, integrase, transposase, Holliday junction resolvase, and Argonaute. These enzymes require divalent metal-ions for activity and share a similar alphabeta-fold in their catalytic domains which brings together three or four conserved acidic residues to bind the metal-ions and form the enzyme active site. Small molecules containing a diketo acid-like moiety are potent inhibitors of HIV integrase and are thought to disrupt catalysis by metal-ion chelation at the active site. These studies suggest similar inhibitors could be found against poxvirus resolvase.;In this work we studied the poxvirus resolvase enzymes encoded for by vaccinia virus and fowlpox virus. We found that the two enzymes display similar DNA cleavage properties, but that the purified vaccinia enzyme was insoluble and not well suited for more detailed analyses. We identified the fowlpox enzyme as a biochemically tractable poxvirus resolvase, which was well suited for basic kinetic analysis, DNA binding studies, and importantly, high throughput screening. We interrogated the DNA binding and cleavage activities of the enzymes using a combination of different DNA substrates, amino acid substitutions, divalent metal-ion conditions, and over 133,000 small molecules. The results suggest that poxvirus resolvase may play a general DNA debranching role during the course of viral replication and that small molecules with diketo acid-like pharmacophores could be developed into selective inhibitors that target resolvase inside poxvirus infected cells. |
