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

¹ Department of Microbiology, University of Guelph, Guelph, ON, Canada, N1G 2W1
² Great Lakes Forestry Centre, Sault Ste Marie, ON, Canada, P6A 2E5

Correspondence
Peter J. Krell
pkrell{at}uoguelph.ca

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'-³²P-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 MgCl₂, 2 mM dithiothreitol and 0·1 mg BSA ml^–1.

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