Choristoneura fumiferana nucleopolyhedrovirus encodes a functional 3′–5′ exonuclease

The Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III 3'-5' exonucleases. The CfMNPV v-trex ORF was cloned into the Bac-to-Bac baculovirus expression-vector system, expressed in insect Sf21 cells with an N-terminal His tag and purified to homogeneity by using Ni-NTA affinity chromatography. Biochemical characterization of the purified V-TREX confirmed that this viral protein is a functional 3'-5' exonuclease that cleaves oligonucleotides from the 3' end in a stepwise, distributive manner, suggesting a role in proofreading during viral DNA replication and DNA repair. Enhanced degradation of a 5'-digoxigenin- or 5'-(32)P-labelled oligo(dT)(30) substrate was observed at increasing incubation times or increased amounts of V-TREX. The 3'-excision activity of V-TREX was maximal at alkaline pH (9.5) in the presence of 5 mM MgCl(2), 2 mM dithiothreitol and 0.1 mg BSA ml(-1).