Virus morphogenesis in the cell: methods and observations

Abstract

Viruses carry out many of their activities inside cells, where they synthesise proteins that are not incorporated into viral particles. Some of these proteins trigger signals to kidnap cell organelles and factors which will form a new macro-structure, the virus factory, that acts as a physical scaffold for viral replication and assembly. We are only beginning to envisage the extraordinary complexity of these interactions, whose characterisation is a clear experimental challenge for which we now have powerful tools. Conventional study of infection kinetics using virology, biochemistry and cell biology methods can be followed by genome-scale screening and global proteomics. These are important new technologies with which we can identify the cell factors used by viruses at different stages in their life cycle. Light microscopy, electron microscopy and electron tomography, together with labelling methods for molecular mapping in situ, show immature viral intermediates, mature virions and recruited cell elements in their natural environment. This chapter describes how these methods are being used to understand the cell biology of viral morphogenesis and suggests what they might achieve in the near future.

Fig. 14.1

Cell organelles used for viral morphogenesis. The cell nucleus is used by herpes- and papillomaviruses; corona-, bunya- and flaviviruses use components of the secretory pathway such as the endoplasmic reticulum and Golgi; togaviruses and the human cytomegalovirus use components of the endo-lysosomal pathway such as endosomes, multivesicular bodies and lysosomes; the African swine fever virus (ASFV) and poxviruses assemble in aggresome-like structures; assembly sites of retroviruses at the plasma membrane might be connected with a cytoplasmic factory. Viruses modify endomembranes, recruit mitochondria and cytoskeleton, and create new inter-organelle contacts

Fig. 14.2

Studying virus assembly by light and electron microscopy. (a, b) Immunofluorescence microscopy of BHK-21 (a) and Vero cells (b) infected with a bunyavirus at 10 h p.i. (h.p.i.). Cells were labelled with an antibody specific for one of the viral structural proteins that concentrates at the assembly sites. A single large perinuclear factory is formed in BHK-21 cells, whereas many mini-factories are seen in Vero cells. (c, d) TEM of BHK-21 (c) and Vero cells (d) at 10 h.p.i. In both cases, similar spherules, the structures that harbour the RC (arrowheads) [22] and viral intermediates (arrows) are distinguished in Golgi membranes. Scale bars, 100 nm

Fig. 14.3

Serial sections, TEM and 3D reconstructions. (a, b) Summary of the principles and differences between conventional ultra-thin sectioning and oriented serial sectioning. (c) Serial sections in TEM and (d) 3D reconstruction showing the interaction between a spherule, the structure that harbours the RC (white) and a viral particle (blue) in Golgi membranes (beige). Mitochondria are segmented in red and rough endoplasmic reticulum (RER) in yellow. (e) 3D reconstruction of a viral factory from a different cell. In this case, 15 serial sections were used. The Golgi complex has been removed to improve visualisation of RC and viral particles. Scale bar, 100 nm

Fig. 14.4

Mimivirus factory in 2D and 3D by TEM and SEM. (a) 2D views of a virus factory as visualised by TEM, showing virus particles (arrows) at various assembly stages. (b) 3D views of a viral factory within an amoeba cell lysed at 8 h.p.i. and visualised by SEM. (c) SEM of a factory isolated at 8 h.p.i. Viral particles are seen at various assembly stages. The arrow indicates a mature virus particle and the arrowhead, an immature particle. (d) SEM of a virus factory isolated at 10 h.p.i. Only mature viruses can be detected. Scale bars, 500 nm in (a) and (d); 2 μm in (b); 300 nm in (c) (Reproduced with permission from [29])

Fig. 14.5

3D electron tomography of virus assembly. (a) Computational slice and (b) 3D reconstruction of the dengue virus factory as visualised by ET. Nascent viral particles (arrowheads) face the spherules that harbour the RC. (c, d) 2D TEM and 3D ET, respectively, of immature VV particles in the process of assembly from cell membranes (arrows). ET shows how the viral envelope is connected to a collection of open membrane structures and how these membranes contribute to envelope formation. Scale bars, 100 nm in (a) and (b); 250 nm in (c) (Reproduced with permission from [32] (a) and (b), and [33] (d))

Fig. 14.6

Molecular mapping of virus assembly with antibodies and clonable tags. (a) Schemes showing the principles of labelling on thin sections, whole permeabilised cells and intact cells. (b) Immunogold detection on a cryosection from a bunyavirus-infected cell. Cells were labelled with a primary antibody specific for a viral scaffolding protein, followed by secondary antibodies conjugated to 10 nm colloidal gold particles. The protein is detected in Golgi membranes (G) and viral particles (arrow). (c) Immunofluorescence detection of the same scaffolding protein in permeabilised cells. (d) Still image from a video recorded in a fluorescence microscope equipped for live cell imaging. Cells were infected with a recombinant virus that expresses the same scaffolding protein fused with GFP. Scale bar, 100 nm

Fig. 14.7

Virus assembly in cell aggresomes. (a) The factories of the ASFV resemble cell aggresomes, as shown by immunofluorescence. Viral proteins (green) co-localise with DNA (blue) inside vimentin cages (red). (b) TEM of an ASFV factory, showing mitochondria (mi), viral intermediates (arrowheads) and mature viruses (arrows). Scale bar, 250 nm. Images kindly provided by Dr. Germán Andrés (CBMSO-CSIC, Spain)

Fig. 14.8

Various technologies and their integration in the study of virus-cell interactions during viral morphogenesis. See text for a description