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Published on: October 27, 2011
A functional histidine-tagged replication initiator protein: implications for the study of single-stranded DNA virus
Julio C Vega-Arreguín1, Tatiana Timchenko, Bruno Gronenborn
1Institut des Sciences du Végétal, CNRS, Avenue de la Terrasse, Bât. 23, 91198 Gif sur Yvette Cedex, France.
Abstract:
Replication initiation of nanoviruses, plant viruses with a multipartite circular single-stranded DNA genome, is triggered by the master Rep (M-Rep) protein. To enable the study of interactions between M-Rep and viral or host factors involved in replication, we designed oligohistidine-tagged variants of the nanovirus Faba bean necrotic yellows virus (FBNYV) M-Rep protein that allow affinity purification of enzymatically active M-Rep from plant tissue. The tagged M-Rep protein was able to initiate replication of its cognate and other FBNYV DNAs in Nicotiana benthamiana leaf disks and plants. The replicon encoding the tagged M-Rep protein multiplied and moved systemically in FBNYV-infected Vicia faba plants and was transmitted by the aphid vector of the virus. Using the tagged M-Rep protein, we demonstrated the in planta interaction between wild-type M-Rep and its tagged counterpart. Such a tagged and fully functional replication initiator protein will have bearings on the isolation of protein complexes from plants.
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