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.

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Cell entry and egress are essential steps in the viral life cycle that govern pathogenesis and spread. Mammalian orthoreoviruses (reoviruses) are nonenveloped viruses implicated in human disease that serve as tractable models for studies of pathogen–host interactions. In this review we discuss the function of intracellular vesicular transport systems in reovirus entry, trafficking, and egress and comment on shared themes for diverse viruses. Designing strategic therapeutic interventions that impede these steps in viral replication requires a detailed understanding of mechanisms by which viruses coopt vesicular trafficking. We illuminate such targets, which may foster development of antiviral agents.