IDENTIFICATION OF ARTICHOKE MOTTLED CRINKLE VIRUS (AMCV) PROTEINS REQUIRED FOR VIRUS-REPLICATION - COMPLEMENTATION OF AMCV P-33 AND P92 REPLICATION-DEFECTIVE MUTANTS

Mutagenesis of the artichoke mottled crinkle virus (AMCV) genome and c omplementation studies between replication-defective mutants were unde rtaken to identify viral protein(s) essential for AMCV replication, In oculation of Nicotiana benthamiana protoplasts with mutant transcripts revealed that null mutations in ORFs 1 [tA33(-)], 2 [tA92(-)] and 6 [ tA7(-)], as well as an ORF 2 mutation [tA92GED] in the GDD motif of th e 92 kDa protein, the putative replicase, prevented accumulation of de tectable levels of progeny RNA, Conversely, mutations of ORFs 3 [tA41( -)], 4 [tA21(-)] and 5 [tA19(-)] did not substantially affect the accu mulation of AMCV genomic and subgenomic RNAs of both positive and nega tive polarity, Inoculation of N. benthamiana plants with transcripts i mpaired in replication revealed that tA92(-) and tA7(-) mutants lead t o replicating pseudorevertants, Functional analysis of these pseudorev ertants showed that: (i) the double stop codon introduced at the end o f ORF 1 to prevent the translational readthrough of the 92 kDa protein reverted to a single amber, ochre or opal codon, giving rise to viabl e genomes; (ii) the putative 7 kDa protein is not essential for genome viability, although the RNA region spanning ORF 6 plays a role in cis in replication, Finally, the two replication-defective mutants tA33(- ) and tA92(-) complemented when coinoculated to N. benthamiana protopl asts, definitively proving that the 33 kDa protein is essential for to mbusvirus genome replication, Analysis of viral RNAs from the coinfect ion experiments showed that tA92(-) was preferentially amplified over tA33(-).