cis preferential replication of Lettuce infectious yellows virus (LIYV) RNA 1: The initial step in the asynchronous replication of the LIYV genomic RNAs

A series of Lettuce infectious yellows virus (LIYV) RNA 1 mutants was created to evaluate their ability to replicate in tobacco protoplasts. Mutants ΔEcoRI, ΔE-LINK, and Δ1B, having deletions in open reading frames (ORFs) 1A and 1B, did not replicate when individually inoculated to protoplasts or when co-inoculated with wild-type RNA1 as a helper virus. A fragment of the green fluorescent protein (GFP) gene was inserted into the RNA 1 ORF 2 (P34) in order to provide a unique sequence tag. This mutant, P34-GFP TAG, was capable of independent replication in protoplasts. Mutants derived from P34-GFP TAG having frameshift mutations in the ORF 1A or 1B were unable to replicate in protoplasts alone or in trans when co-inoculated with wild-type RNA1 as a helper virus. Taken together, these data strongly suggest that LIYV RNA 1 replication is cis-preferential.