Hints on the biological function of a putative nucleolar localization signal of the Ourmia melon virus coat protein.

Ourmia melon virus, is the type member of a recently characterized plant virus group with a tri-segmented genome: RNA1 encodes for the RdRp, RNA2 for a movement protein (MP) and RNA3 for the coat protein (CP). We have previously shown that an amino terminal fusion of GFP and CP locates into the nucleus and preferentially into the nucleolus. Preliminary analysis showed that the first 11 amino acids of the CP sequence are sufficient for nucleolar specific targeting. We then proceeded to perform small deletions and alanine scanning mutagenesis on selected residues of the putative nucleolar localization signal in the CP sequence. 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 Nicotiana benthamiana is correlated to the ability of the same mutant to accumulate the GFP-CP 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 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, 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.