Localization of red clover necrotic mosaic virus polymerase proteins at the endoplasmic reticulum of living plant cells

The replication of positive strand RNA viruses requires association with host membranes and frequently results in membrane proliferation and rearrangement. The fact that widely divergent virus families share form and function of replication strategies is likely due to common evolutionary origin and suggests that elucidating the reproduction strategies of one virus provides insight to virus life cycles in general.; Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA virus in the Dianthovirus genus. RCNMV encodes N-terminally overlapping proteins of 27 kDa and 88 kDa (p27 and p88). p88 encodes the RNA-dependent RNA polymerase, and while the function of p27 is unclear, it is likely to be part of the RCNMV replication complex.; I have investigated the localization of the RCNMV replicase proteins in plants using green fluorescent protein (GFP) fusions observed in live cells using confocal microscopy. The GFP:p27 and GFP:p88 fusions were expressed in N. benthamiana epidermal cells and protoplasts. GFP:p27 consistently localized to the endoplasmic reticulum (ER) and caused membrane restructuring and proliferation. Fractionation of virus-inoculated leaves demonstrated the association of p27 with isolated ER membranes. Additionally, GFP:p27 co-localized with ER-targeted YFP. GFP:p88 also localized to the ER and co-localized with GFP:p27. GFP:p27 and GFP:p88 were associated with invaginations of the nuclear membrane. These channels of ER crossed the nucleus, giving the appearance of fluorescence within the nucleus, while the signal from GFP:p27/p88 was actually associated with ER.; GFP:p88 co-localized with GFP:p27 in two expression systems. This and the fact that GFP:p27 localized to the cortical ER, the nuclear envelope, and cytoplasmic ER domains in the presence of viral genomes and in the absence of any viral proteins suggests that the two proteins co-localize to the ER during infection. Microscopic observations suggest that the virus remodels ER structure to create membranous bodies containing active viral replication complexes. The fact that both proteins independently accumulated in the ER and perturbed the ER morphology suggests that the proteins function together, as part of a larger replication complex.