Three dimensional imaging of the intracellular assembly of a functional viral RNA replicase complex

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.

The viral replication organelles within cells studied by electron microscopy

“…Since then, many studies have demonstrated the power of these methods. Structures harboring the VRCs such as single-membrane spherules and double membrane vesicles have been studied in detail within the replication organelles of West Nile Virus (Gillespie et al, 2010), HCV (Romero-Brey et al, 2012), coxsackievirus B3 (Limpens et al, 2011), Bunyamwera virus (Fontana et al, 2008), Rubella virus (Fontana et al, 2010), Tombusvirus (Fernández de Castro et al, 2017a), a cardiovirus (Melia et al, 2018) and the human reovirus (Fernández de Castro et al, 2014a;Tenorio et al, 2018). The reovirus replication neoorganelle visualized in 3D with two different methods is shown in Fig.…”

“…Scale bar: 100 nm in mainfield in A; 50 nm in inset in A. Modified from Fernández de Castro, I., Fernández, J.J., Barajas, D., Nagy, P.D., Risco, C., 2017a. Three-dimensional imaging of the intracelular assembly of a functional viral RNA replicase complex.…”

“…8A). 3D maps of the replicase molecules inside replication organelles were obtained by electron tomography (Fernández de Castro et al, 2017a) (Fig. 8B).…”

Cryo-electron tomography reveals novel features of a viral RNA replication compartment

“…These viruses complete their entire replication cycle in the host cell cytoplasm. To generate sites for their genomic RNA replication, positive-strand RNA viruses expand and rearrange host cellular membranes (Fernández de Castro et al, 2017; den Boon et al, 2010; Kovalev et al, 2016; Paul and Bartenschlager, 2013; Paul et al, 2014; Romero-Brey and Bartenschlager, 2014). Such membrane-associated RNA replication sites are thought to concentrate essential viral and host factors, and to organize successive steps for efficient viral RNA synthesis (Atasheva et al, 2010; Neuvonen et al, 2011).…”

Drug repurposing for new, efficient, broad spectrum antivirals

“…Moreover, recent developments in light and electron microscopy are having a significant impact in virology. In particular, live-cell microscopy, super-resolution microscopy, correlative light and electron microscopy (CLEM) and 3D imaging are now fundamental tools for studying virus-cell interactions (Bykov et al, 2016;Fernandez de Castro et al, 2017;Francis and Melikyan, 2018;Risco et al, 2014;Witte et al, 2018). Microscopy is also a powerful tool for studying antivirals.…”