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Journal of Virology, August 2009, p. 7761-7769, Vol. 83, No. 15
0022-538X/09/$08.00+0     doi:10.1128/JVI.00179-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Evidence that the Linker between the Methyltransferase and Helicase Domains of Potato Virus X Replicase Is Involved in Homologous RNA Recombination {triangledown}

Heidrun-Katharina Draghici1,{dagger} and Mark Varrelmann1,2*,{dagger}

Department of Crop Sciences, Section Plant Virology, University of Göttingen, Grisebachstrasse 6, D-37077 Göttingen, Germany,1 Institute of Sugar Beet Research, Department of Phytopathology, Holtenser Landstr. 77, D-37079 Göttingen, Germany2

Received 25 January 2008/ Accepted 7 May 2009

Recombination in RNA viruses, one of the main factors contributing to their genetic variability and evolution, is a widespread phenomenon. In this study, an in vivo assay to characterize RNA recombination in potato virus X (PVX), under high selection pressure, was established. Agrobacterium tumefaciens was used to express in Nicotiana benthamiana leaf tissue both a PVX isolate labeled with green fluorescent protein (GFP) containing a coat protein deletion mutation ({Delta}CP) and a transcript encoding a functional coat protein +3'-ntr. Coexpression of the constructs led to virus movement and systemic infection; reconstituted recombinants were observed in 92% of inoculated plants. Similar results were obtained using particle bombardment, demonstrating that recombination mediated by A. tumefaciens was not responsible for the occurrence of PXC recombinants. The speed of recombination could be estimated by agroinfection of two PVX mutants lacking the 3' and 5' halves of the genome, respectively, with an overlap in the triple gene block 1 gene, allowing GFP expression only in the case of recombination. Ten different pentapeptide insertion scanning replicase mutants with replication abilities comparable to wild-type virus were applied in the different recombination assays. Two neighboring mutants affecting the linker between the methyltransferase and helicase domains were shown to be strongly debilitated in their ability to recombine. The possible functional separation of replication and recombination in the replicase molecule supports the model that RNA recombination represents a distinct function of this protein, although the underlying mechanism still needs to be investigated.

* Corresponding author. Mailing address: Institute of Sugar Beet Research, Department of Phytopathology, Holtenser Landstr. 77, D-37079 Göttingen, Germany. Phone: 49 551 5056270. Fax: 49 551 5056299. E-mail: varrelmann{at}ifz-goettingen.de

{triangledown} Published ahead of print on 13 May 2009.

{dagger} H.-K.D. and M.V. contributed equally to this study.

Journal of Virology, August 2009, p. 7761-7769, Vol. 83, No. 15
0022-538X/09/$08.00+0     doi:10.1128/JVI.00179-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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