Virus RNA recombination, one of the main factors for genetic variability and evolution, is thought to be based on different mechanisms. Here, the recently described potato virus X (PVX) recombination assay [Draghici, H.-K. & Varrelmann, M. (2009). 83, 7761–7769] was applied to characterize structural parameters of recombination. The assay uses an -mediated expression system incorporating a PVX green fluorescent protein (GFP)-labelled full-length clone. The clone contains a partial coat protein (CP) deletion that causes defectiveness in cell-to-cell movement, together with a functional CP+3′ non-translated region (ntr) transcript, in leaf tissue. The structural parameters assessed were the length of sequence overlap, the distance between mutations and the degree of sequence similarity. The effects on the observed frequency of reconstitution and the composition of the recombination products were characterized. Application of four different type X intact PVX CP genes with variable composition allowed the estimation of the junction sites of precise homologous recombination. Although one template switch would have been sufficient for functional reconstitution, between one and seven template switches were observed. Use of PVX–GFP mutants with CP deletions of variable length resulted in a linear decrease of the reconstitution frequency. The critical length observed for homologous recombination was 20–50 nt. Reduction of the reconstitution frequency was obtained when a phylogenetically distant PVX type Bi CP gene was used. Finally, the prediction of CP and 3′-ntr RNA secondary structure demonstrated that recombination-junction sites were located mainly in regions of stem–loop structures, allowing the recombination observed to be categorized as similarity-assisted.


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vol. , part 2, pp. 552–562

CLUSTAL_X alignment of PVX-UK3 and different PVX CP gene sequences.

CLUSTAL_X alignment of PVX-UK3 and PVX-4 CP gene plus 3′-ntr compared with 15 recombinant CP genes (Rec1–Rec15).

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