The role of the p33:p33/p92 interaction domain in RNA replication and intracellular localization of p33 and p92 proteins of Cucumber necrosis tombusvirus

Replication of plus-stranded RNA viruses is performed by the viral replicase complex, which, together with the viral RNA, must be targeted to intracellular membranes, where replication takes place in membraneous vesicles/spherules. Tombusviruses code for two overlapping replication proteins, the p33 auxiliary protein and the p92 polymerase. Using replication-competent fluorescent protein-tagged p33 of Cucumber necrosis virus (CNV), we determined that two domains affected p33 targeting to peroxisomal membranes in yeast: an N-proximal hydrophobic trans-membrane sequence and the C-proximal p33:p33/p92 interaction domain. On the contrary, only the deletion of the p33:p33/p92 interaction domain, but not the trans-membrane sequence, altered the intracellular targeting of p92 protein in the presence of wt p33 and DI-72(+) RNA. Moreover, unlike p33, p92 lacking the trans-membrane sequence was still functional in supporting the replication of a replicon RNA in yeast, whereas the p33:p33/p92 interaction domain in both p33 and p92 was essential for replication. In addition, p33 was also shown to facilitate the recruitment of the viral RNA to peroxisomal membranes and that p33 is colocalized with (+) and (-)-stranded viral RNAs. Also, FRET and pull-down analyses confirmed that p33 interacts with other p33 molecules in yeast cells. Based on these data, we propose that p33 facilitates the formation of multimolecular complexes, including p33, p92, viral RNA, and unidentified host factors, which are then targeted to the peroxisomal membranes, the sites of CNV replication.