Asynchronous accumulation of Lettuce infectious yellows virus RNAs 1 and 2and identification of an RNA 1 trans enhancer of RNA 2 accumulation

Time course and mutational analyses were used to examine the accumulation i n protoplasts of progeny RNAs of the bipartite Crinivirus, Lettuce infectio us yellow virus (LIYV; family Closteroviridae). Hybridization analyses show ed that simultaneous inoculation of LIYV RNAs 1 and 2 resulted in asynchron ous accumulation of progeny LIYV RNAs, LIYV RNA 1 progeny genomic and subge nomic RNAs could be detected in protoplasts as early as 12 h postinoculatio n (p.i.) and accumulated to high levels by 24 h p.i. The LIYV RNA 1 open re ading frame 2 (ORF 2) subgenomic RNA was the most abundant of all LIYV RNAs detected. In contrast, RNA 2 progeny were not readily detected until ca, 3 6 h p.i. Mutational analyses showed that in-frame stop codons introduced in to five of seven RNA 2 ORFs did not affect accumulation of progeny LTYV RNA 1 or RNA 2, confirming that RNA 2 does not encode proteins necessary for L IYV RNA replication. Mutational analyses also supported that LIYV RNA 1 enc odes proteins necessary for replication of LIYV RNAs 1 and 2. A mutation in troduced into the LIYV RNA 1 region encoding the overlapping ORF IB and ORF 2 was lethal. However, mutations introduced into only LIYV RNA 1 ORF 2 res ulted in accumulation of progeny RNA 1 near or equal to wild-type RNA 1. In contrast, the RNA 1 ORF 2 mutants did not efficiently support the trans ac cumulation of LIYV RNA 2, Three distinct RNA 1 ORF 2 mutants were analyzed and all exhibited a similar phenotype for progeny LIYV RNA accumulation. Th ese data suggest that the LIYV RNA 1 ORF 2 encodes a bans enhancer for RNA 2 accumulation.