Reovirus σNS and μNS Proteins Remodel the Endoplasmic Reticulum to Build Replication Neo-Organelles

Abstract

Like most viruses that replicate in the cytoplasm, mammalian reoviruses assemble membranous neo-organelles called inclusions that serve as sites of viral genome replication and particle morphogenesis. Viral inclusion formation is essential for viral infection, but how these organelles form is not well understood. We investigated the biogenesis of reovirus inclusions. Correlative light and electron microscopy showed that endoplasmic reticulum (ER) membranes are in contact with nascent inclusions, which form by collections of membranous tubules and vesicles as revealed by electron tomography. ER markers and newly synthesized viral RNA are detected in inclusion internal membranes. Live-cell imaging showed that early in infection, the ER is transformed into thin cisternae that fragment into small tubules and vesicles. We discovered that ER tubulation and vesiculation are mediated by the reovirus σNS and μNS proteins, respectively. Our results enhance an understanding of how viruses remodel cellular compartments to build functional replication organelles.

The viral replication organelles within cells studied by electron microscopy

“…Live-cell imaging has also shown the transformation of cell compartments induced by viruses to build their replication organelles. This is the case of the enterovirus coxsackievirus B3 that disassembles the Golgi complex (Van der Schaar et al, 2016) or the human reovirus that remodels the endoplasmic reticulum to build viral inclusions (Tenorio et al, 2018). New probes for imaging viral RNA will be powerful tools to study viral replication and assembly in live cells (Alonas et al, 2016).…”

“…To identify a rare fluorescently labeled event in a culture of infected cells the most straightforward approach is to grow them on a support that has coordinates and can be imaged by LM, allowing to identify the location of the cell. This support can be photoetched glass coverslips or gridded plastic dishes for conventional preparation, sapphire discs with a carbon pattern for high pressure freezing (HPF), or a finder grid for plunge freezing (Madela et al, 2014;Santarella-Mellwig et al, 2018;Tenorio et al, 2018). The pattern on the substrate allows to localize the cell of interest by phase contrast.…”

“…Viral and cell factors required for spherule biogenesis were also studied by CLEM (Kallio et al, 2013;Spuul et al, 2011). A combination of live cell imaging and CLEM unveiled that nascent reovirus replication organelles, known as viral inclusions (VI) are made of aggregates of tubules and vesicles originated by remodeling of the peripheral endoplasmic reticulum and that two reovirus non-structural proteins, σNS and μNS, are responsible for ER remodeling (Tenorio et al, 2018). Besides, CLEM has great potential for evaluating compounds to treat viral infections (Berger et al, 2014;García-Serradilla et al, 2019;Lowen et al, 2018;Martínez et al, 2014).…”

“…Localization of viral proteins and cell markers helps to explore the origin of viral replication organelles. Moreover, assays of brome-uridine or brome-deoxiuridine incorporation for RNA or DNA viruses, respectively, in combination with immunogold detection on Tokuyasu cryosections, have been very useful to identify active replication organelles (Besse and Puvion-Dutilleul, 1994;Fontana et al, 2008;Tenorio et al, 2018). An example is shown in Fig.…”

Drug repurposing for new, efficient, broad spectrum antivirals

“…Microscopy is also a powerful tool for studying antivirals. With CLEM, scientists can study events at the level of single cells in culture (Fernandez de Castro et al, 2014;Hellstrom et al, 2015;Tenorio et al, 2018) and infected animals (Lowen et al, 2018). CLEM is starting to show its potential for testing antivirals because changes in viral structures assembled in cells where the treatment was effective and in those where the antiviral drug failed can be now studied in detail (Berger et al, 2014;Lowen et al, 2018;Martinez et al, 2014).…”