Dissecting the putative nucleolar localization signal of the Ourmia melon virus coat protein and hints on its biological function.

Ourmiaviruses were recently characterized molecularly as a plus strand RNA plant virus group with a tri-segmented genome, each segment encoding for only one ORF: RNA1 encoding for an RdRp, RNA2 for a movement protein (MP) and RNA3 for the coat protein (CP). This virus group has no detectable phylogenetic relatedness to any plant or animal virus groups so far characterized. Only the RdRp coding sequence shows some distant relatedness to members of the mycovirus family Narnaviridae. Ourmia melon virus, the type member of the Ourmiaviridae. Its extremely simple genomic organization, the ability to infect two important plant model species, Nicotiana benthamiana and Arabidopsis thaliana, and the existence of infectious cDNA clones make it an interesting model system to study plant-virus interaction. We have previously shown that an amino terminal fusion of GFP and Ourmiavirus CP locates into the nucleus and preferentially into the nucleolus and putative Cajal bodies. Through a bioinformatic approach we have selected a putative nuclear (nucleolar) localization signal in a basic amino acid-rich region at the amino terminus of the CP. Preliminary analysis aimed at defining a minimal sequence sufficient to ensure accumulation of the GFP inside the nucleolus showed that indeed the first 11 amino acids of the CP sequence are sufficient for such targeting. We then proceeded to perform small deletions and alanine scanning mutagenesis on selected residues of the putative nucleolar localization signal. At the same time, each mutant was studied for its biological properties in the context of virus infection. Our small deletion analysis showed that ability to efficiently infect systemically N. benthamiana is correlated to the ability of the same mutant to accumulate GFP fusion inside the nucleolus, irrespective of its ability to form virions. Alanine scanning mutagenesis of a number of charged amino acids inside the putative nucleolar localization signal showed that simultaneous presence of three basic residues changes in position 6, 7 and 10 is sufficient to abolish nucleolar targeting of the GFP-CP fusion. Such mutant, in the context of a viral infection, shows ability to form virions of normal morphology, but in N. benthamiana is somewhat impaired in the ability to maintain the initial systemic infection since the infected tissue recovers; in cucurbit hosts, the same mutant is not able to infect systemically both cucumber and melon plants. Although our analyses seem to point to an association between presence of a nucleolar localization signal and a host specific ability to infect systemically (or to maintain infection), we still have no direct evidence that the CP localizes into the nucleus during Ourmiavirus infection cycle.