ABSTRACT
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.
Footnotes
Author contributions
I.F.d.C. designed and conducted experiments, analyzed and interpreted data, made the figures. C.R. designed and supervised experiments, interpreted data and wrote the manuscript. J.J.F. supervised 3D reconstructions and image processing work, and analyzed data. D.B. and P.D.N. provided yeast strains and plasmids, and read the manuscript. All authors commented on and approved the manuscript.
Funding
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
