Background

Replication of positive-stranded RNA viruses depends on the remodeling of cellular membranes. Intracellular membranes serve as a structural scaffold for VRC assembly, provide essential lipids and co-factors that modulate the activity of the viral replicase and protect the viral RNA from the antiviral defenses of the host (Miller and Krijnse-Locker, 2008; den Boon et al., 2010; Nagy and Pogany, 2011; Nagy, 2016). The architecture of replication organelles with active VRCs has been observed by electron microscopy. VRCs assemble in single membrane vesicles or ‘spherules’, tubulovesicular cubic membranes, double membrane vesicles (DMV) or planar oligomeric arrays (de Castro et al., 2013). Spherules are often observed in cells infected by RNA viruses. They form by invagination in a variety of organelles and have a narrow opening to the cytosol (den Boon et al., 2010).

Tomato bushy stunt virus (TBSV) is a small (+) RNA virus that belongs to the Tombusviridae, a family of viruses that infect plants. TBSV has recently emerged as a model virus to study viral replication and virus-host interactions using the yeast Saccharomyces cerevisiae as a model host (Nagy and Pogany, 2011). Studies of S. cerevisiae infected with plant viruses have facilitated the identification of numerous factors needed for viral replication (Nagy, 2008). Tombusviruses encode five proteins including two replication proteins, p92pol and p33. p92pol is the RNA-dependent RNA polymerase. The auxiliary protein p33 is an RNA chaperone that facilitates the recruitment of the viral RNA to the site of replication, in the cytosolic face of peroxisome membranes (McCartney et al., 2005; Jonczyk et al., 2007).

Understanding the biogenesis and functional architecture of VRCs in cell membranes is challenging and it requires sophisticated imaging techniques. Transmission electron microscopy (TEM) has contributed to our understanding of the architecture and organization of macromolecular assemblies in cells. However, methods to unambiguously identify proteins within the environment of the cell are lagging behind. In our lab, we have developed a new labeling method named METTEM from MEtal-Tagging Transmission Electron Microscopy. This method uses the metal-binding protein metallothionein (MT) as a genetically clonable tag for electron microscopy (Diestra et al., 2009; Risco et al., 2012). Mouse MT 1 is a small, 61-amino acid protein with 20 cysteine residues that bind gold atoms very efficiently. MT fused to a protein of interest and treated with gold salts, builds an electron-dense gold nanocluster of around 1 nm diameter, easily visualized by electron microscopy (Mercogliano and DeRosier, 2006 and 2007) (Figure 1). METTEM allows identification and localization of intracellular proteins with high specificity and exceptional sensitivity at molecular-scale resolution ( Diestra et al., 2009; Delebecque et al., 2011; Bouchet-Marquis et al., 2012; Risco et al., 2012; Barajas et al., 2014a; de Castro Martin et al., 2017; Fernandez de Castro et al., 2017).


Figure 1. Imaging viral replicase complexes with METTEM. Viral replicase protein is fused with metallothionein (MT) and expressed in yeast cells (Fernandez de Castro et al., 2017). MT-tagged viral replicase molecules assemble VRCs in peroxisome membranes. Cells are incubated with gold salts in vivo and MT-gold-replicase molecules are visualized by electron microscopy.

Here we described a protocol to visualize TBSV replicase molecules in VRCs using METTEM (Figure 2). The combination of this technology with electron tomography allowed us to study the distribution of replicase molecules in the viral replication compartment in three-dimensions (3D). Due to the high sensitivity of the method we could distinguish different states of aggregation of the viral replicase molecules in situ. This methodology can be used to detect any protein of interest in different subcellular locations of bacteria, yeast and mammalian cells. Furthermore, one advantage of this electron microscopy approach is that it can be used to study many different viruses in a variety of cell types by visualizing the MT tag incorporated in either complete viral particles or their proteins. This method has revealed virus-induced structures not seen before, as reported for Rubella virus, Tombusvirus and influenza virus (Risco et al., 2012; de Castro Martin et al., 2017; Fernandez de Castro et al., 2014 and 2017).


Figure 2. Schematic workflow of the protocol. Pre-grown transformed yeast cells are incubated overnight in YPG. Next day viral replication is induced during 24 h at 23 °C. Cells are treated with zymolyase to obtain spheroplasts. Spheroplasts are incubated with gold salts to build nanoclusters in MT tags. Cell pellets are dehydrated and embedded in resin. Serial sections are transferred to EM grids and imaged by TEM.