Background

Viruses recruit and transform cellular compartments to build membranous viral factories or inclusions, where genome replication and particle assembly take place (Miller and Krijnse-Locker, 2008; Sachse et al., 2019). Following genome packaging and assembly, progeny virions leave the host cell. Both enveloped and nonenveloped viruses can induce cell death by lysis, resulting in the release of viral particles. However, viral egress pathways can also be mediated by intracellular organelles that transport new virions to the plasma membrane, allowing them to exit cells without lysis (Roth et al., 2020). Therefore, many viruses use cellular membranous organelles for replication and assembly, as well as for egress and cell-to-cell transmission (Altan-Bonnet, 2017; Bird and Kirkegaard, 2015).

Reoviruses belong to the Reoviridae family, which includes several pathogens of plants, animals, and humans. Reoviruses are nonenveloped, double-stranded RNA viruses that assemble membranous cytoplasmic structures termed VIs. Early in infection, reovirus nonstructural proteins σNS and μNS induce the remodeling of ER membranes to form VIs (Tenorio et al., 2018). Viral genome replication, secondary rounds of transcription, and capsid assembly occur in VIs and, as a result, mature virions as well as empty capsids gather in this network of membranes. Reovirus uses different types of egress mechanisms depending on the cell type and culture conditions. In some types of cultured cells, reovirus infection leads to NF-κB activation, inducing apoptotic signaling and eventually lytic cell death (Danthi et al., 2013); however, reovirus can undergo nonlytic egress in other cell types such as HBMECs (Lai et al., 2013). In nonpolarized HBMECs, progeny virions exit from discrete areas at the basal surface. The detailed study of these regions is difficult because of their low frequency and location at the cell base. Due to these constraints, late steps of reovirus infection, including nonlytic egress, are not well understood.

The use of several imaging techniques in combination allowed us to discover that the reovirus egress pathway is composed of two different membranous elements called SOs and MCs. SOs are modified lysosomes that are recruited to the periphery of VIs during late phases of infection. Only mature genome-containing virions are collected in SOs, whereas empty capsids are absent. The smaller MCs are formed by budding from SOs and transport viral progeny to the plasma membrane (Fernandez de Castro et al., 2020). ET and 3D reconstruction were essential in defining the spatial relationships of the different compartments and elucidating the intracellular reovirus egress pathway (a summary of the protocol is shown in Figure 1).

ET can be used to visualize the structure of viruses, cells, and their constituents in three dimensions. The chosen approach defines the volume that can be analyzed as well as the possible resolution that can be obtained. In TEM-ET, micrographs of the sample are obtained from various orientations by tilting the specimen, and the resulting micrographs are computationally combined to form a 3D volume (Ercius et al., 2015, Hoppe 1974). Developments in hardware and software during recent years have allowed automatization of the image acquisition process. These innovations enable the production of tomograms, which allows appreciation of cellular organelle ultrastructure and inter-organelle connections in unprecedented detail in 3D. The 3D volumes are useful for studies to determine how cellular compartments interact since they display the spatial organization of structures within the volume, whereas a single 2D image provides only a projection of this information. Interestingly, 3D-ET data showed connections between VIs, SOs, and MCs, the three main components of the reovirus egress pathway. Furthermore, we were able to determine the size of the channels that connect the egress compartments (around 90-100 nm in diameter), which could not be established by analysis of ultra-thin sections (50-70 nm). We were also able to observe that viral egress from MCs to the extracellular medium is mediated by membrane fusion events at the plasma membrane. These events were visualized using double-tilt ET, which orients the specimen around two orthogonal axes, resulting in two independent tomograms computed from each tilt series. The two tomograms were aligned with each other and combined to obtain a single tomogram. As a result, a dual-axis tomogram shows better resolution at any orientation in the plane of the specimen than a single-axis series, in which the specimen is tilted about only one axis (Mastronarde 1997).

In this Bio-protocol, we describe the use of 3D-ET to study the reovirus nonlytic egress pathway and define the morphology and connections between the cellular organelles that compose the exit route. This methodology provides great potential to characterize many other cellular pathways in which different organelles are involved and connected as well as the complexity of pathogen-cell interactions.

Figure 1. Electron tomography of the reovirus egress pathway workflow. Sections of reovirus-infected cells were imaged by transmission electron microscopy to discern reovirus exit zones. The structures of interest were captured, and tilt series were obtained. After image processing and reconstruction, the 3D tomogram can be analyzed. Created with BioRender.com. © 2020 Fernández de Castro et al., originally published in J Cell Biol https://doi.org/10.1083/jcb.201910131.