Virus factories: biogenesis and structural design

Abstract

Replication 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.

Figure 1

Transmission electron microscopy of replication organelles. A. TEM of a plant cell infected with turnip yellow mosaic virus (TYMV). Arrows point to viral spherules, the structures that harbour the replication complex (RC), on the periphery of a chloroplast. The arrowhead indicates the neck‐like connection that communicates the spherule with the cytoplasm. B. BHK‐21 cell infected with Bunyamwera virus. Spherules (arrows) are associated with Golgi membranes. The spherule on the left is connected with a tubular structure. C. Tubuloreticular cubic membranes (star) in contact with double membrane vesicles (DMV; asterisks) in a Vero E6 cell infected with the SARS coronavirus. The dashed circle surrounds a group of curved membranes connected with a budding virus (arrow). D. Oligomeric lattice assembled in vitro by the poliovirus RNA‐dependent RNA polymerase in the presence of RNA, visualized by negative staining and TEM. E. Lattice structure adjacent to the RER in a pancreatic acinar cell infected with coxsackievirus. Bars, 100 nm. F. Models showing our interpretation of the structures in the images. A, C, D and E, reproduced with permission from Prod'homme et al. (2001), Goldsmith et al. (2004), Lyle et al. (2002) and Kemball et al. (2010) respectively.

Figure 2

Virus factories in 3D. A–E. Confocal microscopy and 3D TEM of the large factory assembled by Bunyamwera virus in BHK‐21 cells. A and B. Control (A) and infected (B) cells were labelled with antibodies to protein disulfide isomerase (PDI), a marker of the rough endoplasmic reticulum (RER; red) and the viral glycoprotein (Gc; green). Nuclei were stained with DAPI (blue). Confocal microscopy (B) shows massive recruitment of RER elements around the factory. C. Factory as visualized by TEM of serial sections, 3D reconstruction and image processing. Mitochondria (red) surround a network of RER (yellow) and Golgi (beige) membranes. Viral tubes, the structures that harbour the viral RC, are grey. N, nucleus. D and E. Volumes at higher magnification, showing a viral tube attached to Golgi and RER membranes (D), and another near a viral particle (blue) (E). Bars: A and B, 10 μm; E, 100 nm. C–E, reproduced with permission from Fontana et al. (2008). F–I. Mini‐factories of rubella virus in BHK‐21 cells. F. Freeze‐fracture, metal replication and TEM shows the recruitment of mitochondria (mi) and RER around the replication organelle (asterisk), which is a modified lysosome. N, nucleus. G. Three‐dimensional model of the factory as visualized by ET. The replication organelle (yellow) is surrounded by RER (green) and presents an opening to the cytosol (arrow). A mitochondrion is coloured red. H and I. Computational tomographic slices from different tomograms, showing contacts between the RER and the replication organelle (asterisks), seen as closely apposed membranes (arrow in H) and protein bridges (arrow in I). Bars: F, 200 nm; H and I, 50 nm. F–I, reproduced with permission from Fontana et al. (2010). J–O. Viral factories assembled by the giant mimivirus in amoeba cells. J. Electron tomographic slice of an early replication factory at 4 h.p.i. (stage 1). Arrows point to viral cores. K. Surface rendering of the boxed region in J, showing the two viral cores (yellow) surrounded by viral DNA (blue). L. Thin section of a virus factory at 8 h.p.i. (stage 2) as visualized by TEM, revealing viral particles (arrows) at various assembly stages. M. Three‐dimensional views of a viral factory within an amoeba cell lysed at 8 h.p.i. and visualized by SEM. N. SEM of a virus factory isolated at 8 h.p.i. The blue arrow indicates an immature virus particle and the white arrow, a mature particle. O. SEM of a virus factory isolated at 10 h.p.i. (stage 3). Only mature viruses can be detected. Bars: J, 200 nm; L and O, 500 nm; M, 2 μm; N, 300 nm. J, K, reproduced with permission from Mutsafi et al. (2010); L–O, from Zauberman et al. (2008).