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

Abstract

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 (⌬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.RNA virus variability is mainly generated by mutation, reassortment (in case of viruses encoding a multipartite genome), and covalent rearrangements of recombination origin including duplications, deletions, and insertions. Subsequent phenotypic selection results in adaptation to modified environmental conditions (29,85). The mutation rate is high among RNA viruses and driven by infidelity of template replication due to missing proofreading ability of RNA-dependent RNA polymerases (RdRps) (30, 31). As recombination links two or more RNA molecules, it is believed to rescue viral genomes by repairing mutation errors, leading to enhanced viral fitness (2,35,96). Along with mutation, recombination represents the major source of evolutionary variation of plant virus populations (43, 44). RNA recombination in plant viruses is a widespread phenomenon detected in several plant virus groups and families (reviewed in references 1, 5, and 22). Experimental in vivo evidence has been found in several different virus species (3,18,56,76,96). In general approaches for the detection of recombination can be categorized by the selection pressure applied. One frequently used system is the generation of a defective virus mutant and a transgenic plant expressing the corresponding functional gene, thereby applying high selection pressure on the production of functionally re...

RNA-RNA Recombination in Plant Virus Replication and Evolution

“…Recently, the pentapeptide scanning mutagenesis of the PVX replicase indicated that the hydrophilic linker between the N-terminal methyltransferase (MT) and the C-terminal Hel-polymerase (HEL-POL) domains possesses a distinct recombination function vital www.annualreviews.org • RNA Recombination in Plant Viruses for maintenance of genome integrity (37). The pentapeptide insertion in two replicase amino acid positions 430 and 434 interfered with the dynamic interaction necessary for a possible template switch of the molecule.…”

“…During in vitro transcription with purified viral RNA-dependent RNA polymerase (RdRp) and derivatives of viral RNAs, both parental and recombined type products are synthesized. ORFs are marked in green (a,b), noncoding regions are marked with gray lines, and red indicates mutations.Other recombination systems were developed relying on satellite and genomic RNAs of Turnip crinkle virus (TCV)(149), or defective interfering RNAs (DI RNAs) of Tomato bushy stunt virus (TBSV), Cucumber necrosis virus (CNV)(144), and Potato virus X (PVX)(37,38). These systems employed at least two parental RNA molecules with one component carrying a deleterious mutation.…”

The Unfolded Protein Response Is Triggered by a Plant Viral Movement Protein

“…Total protein was extracted from leaves by grinding samples with extraction buffer (4 M urea, 4% SDS, 0.2 M dithiothreitol, 20% glycerol, 0.2 M Tris-HCl, pH 6.8, and 0.04% bromphenol blue; Draghici and Varrelmann, 2009) or standard protein extraction buffer (100 mM Tris-HCl, pH 7.5, 10 mM KCl, 0.4 M Suc, 10% glycerol, and 10 mM phenylmethylsulfonyl fluoride; Sambrook et al, 1989) and quantified using Bradford Reagent (Sigma-Aldrich). Thirty micrograms of protein for each sample was loaded onto a 4% to 20% precast gradient or 10% SDS-PAGE gels (Bio-Rad) and electroblotted to Hybond-P (GE Healthcare) using standard protocols (Sambrook et al, 1989).…”

Replication of Plant Viruses

“…A mutation within the polymerase domain of 2a inhibited heteroduplex-mediated non-HR while increasing homologous crossovers (Figlerowicz et al, 1997). The hydrophilic linker between the N-terminal MT and the C-terminal HELpolymerase (HEL-POL) domains of the PVX replicase possesses a distinct recombination function vital for maintenance of genome integrity (Draghici and Varrelmann, 2009). The hydrophilic linker between the N-terminal MT and the C-terminal HELpolymerase (HEL-POL) domains of the PVX replicase possesses a distinct recombination function vital for maintenance of genome integrity (Draghici and Varrelmann, 2009).…”