Evidence for similarity-assisted recombination and predicted stem-loop structure determinant in potato virus X RNA recombination.

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 in vivo potato virus X (PVX) recombination assay [Draghici, H.-K. & Varrelmann, M. (2009). J Virol 83, 7761-7769] was applied to characterize structural parameters of recombination. The assay uses an Agrobacterium-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 Nicotiana benthamiana 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.

Evidence that the linker between the methyltransferase and helicase domains of potato virus X replicase is involved in homologous RNA recombination.

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 (DeltaCP) 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.

Functional mapping of PVX RNA-dependent RNA-replicase using pentapeptide scanning mutagenesis-Identification of regions essential for replication and subgenomic RNA amplification.

The replicase protein of Potato virus X (PVX), type species of the genus Potexvirus, was selected to identify regions essential for replication and subgenomic RNA synthesis. Replicase amino acid (aa) sequence alignment of 16 Potexvirus species resulted in the detection of overall sequence homology of 34.4-65.4%. Two regions of consensus with a high proportion of conserved aa (1-411 and 617-1437 according to PVX) were separated by a hyper-variable linker region. Pentapeptide scanning (PS) mutagenesis in a PVX full-length clone expressing green fluorescent protein (GFP) was carried out. For 69 selected PS-mutants where insertions were spread randomly over the replicase ORF the position of the insertion was determined. The replication activity was evaluated by GFP expression from subgenomic viral RNA of PVX replicase mutants. Only one functional PS-mutant was detected in the N-terminal 430 aa, containing the conserved methyltransferase domain of the protein. In the linker region from aa 430-595, nine mutations were discovered which did not induce significant effects on the replicase replication ability. The part of the protein including helicase and polymerase domains was highly intolerant for the PS insertion as demonstrated by 24 independent more or less uniformly spread mutants.