Positive-strand RNA viruses, which can be devastating pathogens in humans, animals and plants, replicate their genomes on intracellular membranes. Here, we describe the three-dimensional ultrastructural organization of a tombusvirus replicase in yeast, a valuable model for exploring virus–host interactions. We visualized the intracellular distribution of a viral replicase protein using metal-tagging transmission electron microscopy, a highly sensitive nanotechnology whose full potential remains to be developed. These three-dimensional images show how viral replicase molecules are organized when they are incorporated into the active domains of the intracellular replication compartment. Our approach provides a means to study protein activation mechanisms in cells and to identify targets for new antiviral compounds.
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This work was supported by the Spanish Ministry of Economy and Competitiveness [grant numbers BIO2012-33314 to C.R. and TIN2012-37483 to J.J.F.]; and the US National Science Foundation [grant number MCB-1122039 to P.D.N.].
Data availability: Supplementary material movies are available at https://dx.doi.org/10.6084/m9.figshare.3159094.v1
Supplementary information: Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.181586.supplemental
Fernández de Castro, Isabel; Fernández, José J.; Barajas, Daniel; Nagy, Peter D.; and Risco, Cristina, "Three-Dimensional Imaging of the Intracellular Assembly of a Functional Viral RNA Replicase Complex" (2017). Plant Pathology Faculty Publications. 64.