Analysis of sequences in the Cymbidium ringspot virus replicase protein p33 that promote targeting and anchoring to endoplasmic reticulum membranes

RNA replication of Cymbidium ringspot virus (CymRSV) normally occurs in plant and yeast cells in association with membranes derived from peroxisomes. However, in yeast strains devoid of these organelles CymRSV RNA replication takes place on the perinuclear membrane and cytoplasmic endoplasmic reticulum (ER) strands. The association of the CymRSV RNA replication complex to ER is mediated by the ORF 1- encoded protein p33. To analyze which part of this protein contains the information leading to localization to ER, a deletion analysis was carried out. The behaviour of deletion mutants was studied by confocal immunofluorescence microscopy and membrane floatation gradient analysis. It was shown that the signal targeting p33 to ER membranes largely coincides with the peroxisomal targeting signal, but contains additional elements in the first 87 amino acids.