The Loss of Infectivity Due to a 1 nt-Deletion in the RdRP Coding Region of an Ourmiavirus Agroclone Can Be Restored by the Presence of a Specific Mutation in a Distinct Region of the Viral RdRP.

Ourmiaviruses possess a compact three-segmented positive strand ssRNA genome encoding for only three ORFs: the viral RdRP (RNA1), a movement protein (RNA2) and the CP (RNA3). This genome organization possibly originated through re-assortment between a fungal virus of the Narnaviridae family, and a plant virus. Two sets of three agroclones representing each genomic RNA, were assembled. Upon inoculation one set caused only mild symptoms on Nicotiana benthamiana plants, whereas the other caused the typical necrotic phenotype. Analysis of reassorted genomic segments between the two sets allowed us to map the necrotic phenotype to the RNA1. Sequence comparison between the two RNA1variants revealed 17 mutations, three of which resulted in amino acid substitutions. Surprisingly the mild infectious variant also carried a 1 nt-deletion in the RdRP coding sequence, putatively resulting in the premature interruption of protein synthesis. Nevertheless, infectivity was observed in vivo and the RdRP coding sequence without the deletion was present in the RNA progeny of systemically infected plants. Analysis of the chimeric agroclones obtained exchanging three DNA segments spanning regions of the RNA1 (segment I, II and III respectively) revealed that the determinant for the necrotic phenotype is located in segment II. Site directed mutagenesis was performed on the codons of the three amino acid substitutions and unequivocally identified the G1104A mutation in segment II as the only one responsible for the necrotic phenotype. By contrast, the 1 nt deletion present in segment I of the mild variant unexpectedly had no role. The chimeric analysis revealed a second surprising feature: although both original clones were fully infectious, some of the chimeras were not. In particular, each of the chimeric clones combining the segment I with 1 nt-deletion and the segment III from the necrotic phenotype were not infectious in N. benthamiana plants. This is the first report of an infectious clone carrying a single nucleotide deletion in the region encoding for the viral RdRP; previously a 1nt insertion was shown to be present in Citrus tristeza virus infectious clones. We also provide the first evidence that the ability to restore the deletion through a 1nt insertion is genetically determined in a specific region of the viral RdRP.