SummaryReplication and assembly of many viruses occur in specific intracellular compartments known as 'virus factories'. Our knowledge of the biogenesis and architecture of these unique structures has increased considerably in the last 10 years, due to technical advances in cellular, molecular and structural biology. We now know that viruses build replication organelles, which recruit cell and viral components in a macrostructure in which viruses assemble and mature. Cell membranes and cytoskeleton participate in the biogenesis of these scaffolds and mitochondria are present in many factories, where they might supply energy and other essential factors. New inter-organelle contacts have been visualized within virus factories, whose structure is very dynamic, as it changes over time. There is increasing interest in identifying the factors involved in their biogenesis and functional architecture, and new microscopy techniques are helping us to understand how these complex entities are built and work. In this review, we summarize recent findings on the cell biology, biogenesis and structure of virus factories.

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.

Bunyamwera virus (BUNV), which belongs to the genus Orthobunyavirus, is the prototypical virus of the Bunyaviridae family. Similar to other negative-sense single-stranded RNA viruses, bunyaviruses possess a nucleocapsid protein (NP) to facilitate genomic RNA encapsidation and virus replication. The structures of two NPs of members of different genera within the Bunyaviridae family have been reported. However, their structures, RNA-binding features, and functions beyond RNA binding significantly differ from one another. Here, we report the crystal structure of the BUNV NP-RNA complex. The polypeptide of the BUNV NP was found to possess a distinct fold among viral NPs. An N-terminal arm and a C-terminal tail were found to interact with neighboring NP protomers to form a tetrameric ringshaped organization. Each protomer bound a 10-nt RNA molecule, which was acquired from the expression host, in the positively charged crevice between the N and C lobes. Inhomogeneous oligomerization was observed for the recombinant BUNV NP-RNA complex, which was similar to the Rift Valley fever virus NP-RNA complex. This result suggested that the flexibility of one NP protomer with adjacent protomers underlies the BUNV ribonucleoprotein complex (RNP) formation. Electron microscopy revealed that the monomer-sized NP-RNA complex was the building block of the natural BUNV RNP. Combined with previous results indicating that mutagenesis of the interprotomer or protein-RNA interface affects BUNV replication, our structure provides a great potential for understanding the mechanism underlying negative-sense single-stranded RNA RNP formation and enables the development of antiviral therapies targeting BUNV RNP formation.assembly | packaging B unyaviruses constitute the largest segmented negative-sense single-stranded RNA (-ssRNA) virus family, which is subdivided into Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus genera (1). The genomes of Bunyaviridae members are featured by three segments, the large (L), middle (M), and small (S) segments (2, 3). The L segment encodes an RNA-dependent RNA polymerase (RdRp), the M segment encodes a precursor of glycoproteins (Gn and Gc), and the S segment encodes a nucleocapsid protein (NP). Moreover, a few Bunyaviridae members possess a nonstructural protein (NSs and/or NSm) using an ambisense coding strategy by the S and M segments (4).Similar to other -ssRNA viruses, all members of the Bunyaviridae family contain a ribonucleoprotein (RNP) complex composed of genomic RNA enwrapped by the NP (4). After entry into the cytoplasm through membrane fusion mediated by glycoproteins, the RNP is released from the virion and serves as the template with which the copackaged RdRp transcribes mRNAs from the viral genome in the RNP. In the later stage of virus replication, complementary positive-strand RNA (cRNA) is produced in the form of an RNP. The RNP serves as the template for replication that generates the viral genomic RNA in the form of an RNP ready to be packaged in the virion. Throughout...

Most viruses that replicate in the cytoplasm of host cells form neo-organelles that serve as sites of viral genome replication and particle assembly. These highly specialized structures concentrate viral replication proteins and nucleic acids, prevent the activation of cell-intrinsic defenses, and coordinate the release of progeny particles. Despite the importance of inclusion complexes in viral replication, there are key gaps in the knowledge of how these organelles form and mediate their functions. Reoviruses are nonenveloped, double-stranded RNA (dsRNA) viruses that serve as tractable experimental models for studies of dsRNA virus replication and pathogenesis. Following reovirus entry into cells, replication occurs in large cytoplasmic structures termed inclusions that fill with progeny virions. Reovirus inclusions are nucleated by viral nonstructural proteins, which in turn recruit viral structural proteins for genome replication and particle assembly. Components of reovirus inclusions are poorly understood, but these structures are generally thought to be devoid of membranes. We used transmission electron microscopy and three-dimensional image reconstructions to visualize reovirus inclusions in infected cells. These studies revealed that reovirus inclusions form within a membranous network. Viral inclusions contain filled and empty viral particles and microtubules and appose mitochondria and rough endoplasmic reticulum (RER). Immunofluorescence confocal microscopy analysis demonstrated that markers of the ER and ER-Golgi intermediate compartment (ERGIC) codistribute with inclusions during infection, as does dsRNA. dsRNA colocalizes with the viral protein σNS and an ERGIC marker inside inclusions. These findings suggest that cell membranes within reovirus inclusions form a scaffold to coordinate viral replication and assembly.