Virus Morphogenesis in the Cell: Methods and Observations
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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.
KeywordsVirus factory Virus assembly Virus morphogenesis Genomics Proteomics Light microscopy Electron microscopy Tomography 3D electron microscopy Super-resolution microscopy Correlative microscopy Molecular mapping Immunofluorescence Immuno gold Immuno electron microscopy Clonable tags
African swine fever virus
Correlative light and electron microscopy
Endosomal sorting complex required for transport
Green fluorescent protein
Hepatitis C virus
Human immunodeficiency virus
Scanning electron microscopy
Transmission electron microscopy
14.1 Introduction: Cell Biology of Virus Morphogenesis and the Concept of the Virus Factory
Virus factories are usually rather large, several microns in diameter, and are very dynamic, as their structure changes over time in accordance with virus needs . This strategy appears to allow viruses to generate progeny with maximum efficiency in the use of cell resources. It is also thought that viruses can use these scaffolds to protect themselves from cellular antiviral defences . Viral factories were first described for large DNA viruses such as the African swine fever virus (ASFV) and the poxviruses; we now know that many DNA and RNA viruses build factories in the cytosol, in the nucleus, or both. Due to our limited knowledge of cell nucleus organisation, characterisation of nuclear viral factories is only beginning. In contrast, viral factories assembled in the cytosol are being studied extensively and there are many reports on their architecture and activities. Inside these cytosolic factories, morphogenesis of new viruses begins with the transport of replicated genomes from the structures that harbour the replication complexes (RC) to the assembly sites. Depending on the virus, a number of maturation steps will follow (see Chap. 13 for details) before new viral particles are ready for delivery and propagation.
This chapter will describe fundamental tools for diving inside the infected cell and understanding virus assembly. We also describe relevant examples of virus-cell interactions during virus morphogenesis that are being discovered using in situ techniques.
14.2 Methods for the Study of Virus-Cell Interactions During Morphogenesis
By identifying essential cell factors, studies based on recent advances in molecular biology, genomics and proteomics are broadening our knowledge of viral morphogenesis. To confirm and understand the role of potentially interesting genes in viral assembly, the information acquired must be subsequently analysed at a more complex level in infected cells. Exploring existing databases on cell pathways which combine information on genetic, metabolic and signal networks based on the literature can be a first step in further work that on many occasions will also include imaging with a variety of microscopy technologies.
14.2.1 Identification of Essential Cell Factors
Characterisation of infection kinetics is the first step in the study of viral morphogenesis. Conventional virology, biochemistry and cell biology methods allow us to determine optimal experimental conditions, including times post-infection (p.i.) and the most appropriate cell types. Key factors can be identified using two main groups of methods, (1) those that analyze gene expression patterns, and (2) those that study protein-protein interactions.
A number of new techniques can be applied to study interactions between viruses and cells on a genomic scale . Microarrays are being used to identify mRNA transcription patterns in different phases of the virus life cycle. DNA gene chips detect differences in gene expression between uninfected and virus-infected cells and at different infection stages. Gene data bases are then useful for associating the genes identified with specific cell pathways [4, 5]. High-throughput screening based on RNA interference (RNAi) is another category of methods that analyse gene expression patterns. RNAi is an RNA-dependent gene silencing process within living cells that is often exploited to study the function of genes. This emerging technology is used to study how viruses interact with their hosts at the molecular level. Analysis at various times post-infection has identified a number of cell factors potentially involved in viral morphogenesis, for example for dengue , influenza  and retroviruses . RNAi may be used for large-scale screens that systematically shut down each gene in the cell. Studies using this approach have shown a requirement for cell factors such as the ESCRT machinery for assembly of the human immunodeficiency virus, HIV .
The group of techniques termed proteomics includes powerful methods to study protein-protein interactions. The yeast two-hybrid (Y2H) assay system remains one of the most amenable techniques and is widely used to search for virus-host interactions. Y2H works by expressing two candidate proteins in the yeast cell. Bait and prey proteins are fused either to a promoter-specific DNA-binding domain or to a transcription activation domain. Interaction between the two proteins in the yeast nucleus brings both domains together so that they can initiate expression of a reporter gene . Individual bait proteins can be screened for interaction with a library of prey proteins. Genome-scale Y2H studies were used to identify 314 virus-host interactions for HCV  109 interactions for vaccinia virus  and nine for HIV-1 . Similar techniques, such as the yeast three-hybrid system, can be used to study interactions between nucleic acids and proteins [14, 15]. This group of methods also includes pull-down and immunoprecipitation assays, and tandem affinity purification (TAP) tagging approaches, as well as protein identification by quantitative and semi-quantitative mass spectrometry. Whereas Y2H usually detects transient interactions, affinity-tag purification mass spectrometry shows stable, stoichiometric complexes. Since cell proteins often incorporate into viral particles, these techniques can be applied to the study of protein-protein interactions in the infected cell and in purified viral assembly intermediates . This is the case of clathrin, for example, which was found in retrovirus particles; clathrin was only recently identified as one of the cell factors that facilitate accurate morphogenesis of several retroviruses . Y2H technology also detected cell proteins that interfere with virus assembly and viral proteins that block them. This is the case of tetherin, first detected by proteomics and mass spectrometric protein identification as a cell factor that restricts retrovirus assembly , and later confirmed as a restriction factor for a wide variety of enveloped viruses . Viruses have several anti-tetherin proteins to counteract the effect of this factor . The current challenge of high-throughput technologies is to develop more efficient informatics tools to accurately analyse the vast amount of information they can provide .
The next step in the characterisation of in-cell virus assembly pathways would be to study the specific roles of the factors identified in their natural environment; to do this, we must of course return to the infected cell. Functions can be tested by protein depletion or overexpression, by mutagenesis or by protein targeting with tags. To visualise key factors in infected cells, there are a variety of classical and novel microscopy techniques that will be described in the following sections.
14.2.2 Studying Viral Morphogenesis in Situ with Light and Electron Microscopy
Electron microscopy generally uses ultra-thin sections of cells previously embedded in plastic resins after conventional fixation and dehydration; alternatively, cells are processed at low temperature after or upon fixation for optimal preservation of ultrastructure (see Chap. 3 for sample preparation details), or subjected to criofixation procedures such as high-pressure freezing prior to freeze substitution and embedding, or freeze-fracture analyses. With resolutions in the range of a few nanometers, cell TEM can show changes in shape and size of virus assembly intermediates in specific intracellular compartments [25, 26] (Fig. 14.2c, d). TEM of infected cells shows that mitochondria, endomembranes and cytoskeleton often participate in organising the structure that supports viral assembly. To complete maturation and become infectious, immature viral particles must often travel within the factory to find specific cell factors. At late times post-infection, once virus progeny have been produced and must find their way out of the cell, the factory can be dismantled .
TEM of cells in two dimensions provides a first glimpse of the viral morphogenetic pathways in situ. The size of cells (several microns in diameter) and the thickness of ultra-thin sections (50–100 nm) nonetheless constitute a major limitation, because we are actually studying single planes of much larger structures. In these conditions, many elements and contacts can be missed. Three-dimensional TEM avoids this limitation; the variety of methods available is described in the following sections.
14.2.3 Visualising Virus Assembly in Three Dimensions I: 3D Reconstructions of Serial Sections, TEM and SEM
TEM in combination with freeze-fracture and metal shadowing allows higher resolution 3D views of viruses inside intact cells. Frozen cells are fractured mechanically and covered with a thin layer of platinum and carbon in a vacuum chamber, the metal replicas are then washed to eliminate organic materials and mounted on EM grids. The main limitation of this approach derives from the unpredictable patterns of the fracture planes and the difficulty in interpreting the images. It can nevertheless be useful when studying the morphogenesis of large enveloped viruses ( Chap. 11) and their interactions with cell endomembranes during assembly and maturation .
14.2.4 Visualizing Virus Assembly in Three Dimensions II: Electron Tomography
In summary, two main groups of methods are available for 3D studies of viral assembly. 3D reconstructions of serial sections by TEM or analysis of surface morphology by SEM will show the general organisation of the virus factory, the inter-organelle contacts, and changes in the cell compartments where viruses are formed. In contrast, electron tomography is more appropriate for studying individual viral particles and to obtain fine details of their maturation in situ.
14.3 Molecular Mapping of Viral Morphogenesis
The discipline known as histochemistry includes a great variety of techniques to visualise molecules in biological samples . There are several histochemical methods to localise nucleic acids, lipids, sugars, and other molecules, some of which have been used to label viruses in cells. Nonetheless, protein-labelling techniques are by far the most developed; because of their importance in the study of virus morphogenesis, in this section we will focus on methods that detect proteins in light and electron microscopy.
14.3.1 Antibodies, Immunofluorescence and Immunoelectron Microscopy
Antibodies detect proteins with high specificity and variable sensitivity. Primary antibodies generated against proteins of interest are detected with secondary antibodies conjugated to a fluorescent probe for LM visualisation, or to an electron-dense colloidal gold particle, which is easy to detect by EM in immunogold assays (Fig. 14.6a). Antibodies have been and are still fundamental tools in electron microscopy [36, 37]. When immunolabelling proteins on cryosections, where cells have not been dehydrated and maintain their proteins in a natural hydrated state, the sensitivity of antibody detection is usually higher than that obtained when labelling sections of dehydrated, resin-embedded cells. Since the introduction of cryosectioning, the method has been improved and perfected [38, 39]. This approach allows colocalisation of nascent and maturing viral particles in specific cell compartments and the proteins being incorporated into assembling virus particles (Fig. 14.6b). Although antibodies are usually very specific, information derived from these experiments is later confirmed in biochemical assays.
Due to their large size, antibodies that recognise internal structures must be used on cell sections. Alternatively, cells can be permeabilised to label intracellular compartments (Fig. 14.6c), although this is incompatible with preservation of fine ultrastructure. Certain permeabilisation protocols use the bacterial exotoxin streptolysin O (SLO) to open pores in the plasma membrane while leaving intracellular membranes untouched. This was used in pre-embedding immunogold assays to follow viral and cell proteins during VV assembly from intracellular membranes .
An important limitation of antibodies is their variable sensitivity due to epitope loss during sample preparation and to macromolecular interactions inside cells that often mask the protein epitopes in vivo. This is particularly problematic when the proteins of interest are part of densely packed macromolecular complexes, such as those involved in virus assembly. The use of clonable tags can overcome these limitations.
14.3.2 Clonable Tags
Jellyfish green fluorescent protein (GFP), its mutants and homologues have caused a true revolution in cell biology. If proteins fused to a fluorescent tag maintain their normal functions, their movements and interactions with intracellular structures can be tracked in living cells (Fig. 14.6d). In conjunction with the new generation of LM methods, we can even follow the assembly of individual virus particles at real time . GFP is a relatively large molecule (27 kDa) and, since viral proteins have very strict structural requirements, fusion to GFP can produce loss of function. If this is the case, probes smaller than GFP could be a viable option. The group of smaller probes includes the fluorescent flavoprotein known as miniSOG, which is less than half the size of GFP , or the smaller tetra-cysteine motifs, which are visualised after incubation with fluorescent biarsenical molecules such as ReAsH or FlAsH .
The use of clonable tags in TEM and ET would be a considerable advance for high resolution studies of virus assembly. The first clonable tags for EM validated in cells have yet to be used in virus morphogenesis studies, but show good prospects and will be listed in Sect. 14.5.3.
14.4 The Search for Signalling Pathways
The initiation of virus morphogenesis usually requires major reorganisation of cell membrane compartments and the cytoskeleton. Many laboratories are applying the methods described in previous sections to understand how viruses take control of cells and recruit all the necessary materials for their morphogenesis. The complexity of the interactions inside infected cells can be extraordinary, as illustrated by the viruses known as virophages that parasitise the factory built by other viruses. Some images of cells co-infected by the giant mimivirus and the virophage “Sputnik” suggest that the small virophage not only uses the materials recruited by the mimivirus for its own replication, but might even hide inside the mimivirus particles to exit the cell . We still do not know how organelles and materials are recruited or how replicated viral genomes are transported from RC to assembly sites. We do know, however, that viruses target their proteins to specific cell compartments, that some viruses use a cellular defence mechanism termed the aggresome response, and that signals related to organelle movement on cytoskeletal tracks could also be involved [1, 2].
14.4.1 DNA Viruses and Cell Aggresomes
A similar strategy might be used by viruses that replicate and assemble in the cell nucleus and associate with structures known as POD (potential oncogenic domains). POD are nuclear aggresomes used by herpes-, papilloma-, adeno- and parvoviruses. Recent observations suggest that some RNA viruses use aggresomes to build their factories. Since the common feature of factories built by RNA viruses is the remodelling and recruitment of cell endomembranes, however, different signalling pathways must be triggered in this case.
14.4.2 RNA Viruses and Membrane Remodelling
DNA viruses usually build distinct, and even distant factories for genome replication and morphogenesis. Herpesviruses, for example, must connect the first steps of assembly inside the nucleus with subsequent incorporation of proteins and membranes in the cytosol; poxviruses must coordinate replication in cytosolic mini-nuclei with primary assembly in aggresome-like structures and final wrapping in the Golgi apparatus . In contrast, RNA viruses often induce the construction of a single sophisticated membranous web in which replication complexes and assembly sites are located near each other. Expression of viral replicase complexes is usually sufficient to trigger membrane remodelling and organelle recruitment [47, 48].
Viruses take control of cell endomembranes by interfering with lipid metabolism, protein regulation and transport. The secretory pathway is the most common target for this virus-induced membrane remodelling, while the endocytic pathway also participates in some cases (Fig. 14.1). The two pathways are closely related, in fact, and converge at the trans-Golgi compartment. Described in the literature with many different names, the membranous tubuloreticular structures (TBS) often detected in infected cells are indeed cubic membranes that consist of highly curved, 3D-folded lipid bilayers. Alterations in cholesterol metabolism are linked to the biogenesis of cubic membranes, used for assembly by some viruses such as the SARS (severe acute respiratory syndrome) coronavirus . Other viruses depend on phosphatidylcholine synthesis, fatty acid synthesis or geranylation. The highly curved nature of cubic membranes, possibly induced by multiple interactions between replicase proteins, might assist virus budding. Virus replication often slows secretion, however, which means that viruses can interfere with membrane trafficking proteins and their regulation by small GTPases [2, 47]. Additional host factors involved in virus-induced membrane remodelling include the SNARE (soluble NSF attachment protein receptor) proteins, which are mediators of vesicle fusion used by the human cytomegalovirus (HCMV), the VAMP-associated proteins (VAP) that bind to SNARE and are used by hepatitis C virus, and the ESCRT (endosomal sorting complex required for transport), which are essential for HIV assembly in the plasma membrane [19, 47, 50].
Inside membranous webs, virus assembly is precisely coordinated with transport of replicated viral genomes from the RC; the specific machinery remains to be identified. Lessons from plant viruses and their movement proteins involved in viral genome transport inside cells and between cells through plasmodesmata can provide some clues for the study of animal viruses .
Finally, signalling related to cytoskeleton-mediated organelle movement, particularly in the case of mitochondria, can be essential for factory assembly and virus morphogenesis. Mitochondria attach to membranous webs and are thought to provide energy for viral factory activities. They can have additional roles, as in infected cells it was observed that some mitochondrial proteins abandon the organelle and are integrated into the factories, where they interact with viral proteins [52, 53].
14.5 Perspectives and Conclusions
Some of the following new methodologies have not yet been applied to the study of viral morphogenesis, but their capacity to track macromolecules in cells suggests great potential for the study of virus assembly.
14.5.1 Super-Resolution Fluorescence Microscopy
Limited by the diffraction barrier, conventional light microscopy methods reach resolutions of ~100–500 nm. Many viruses and cell substructures are smaller than this and cannot be solved by LM. In recent years, several laboratories have developed a group of optical microscopy methods, termed super-resolution microscopy, that have improved spatial resolution by an order of magnitude over the diffraction limit . Future improvements in microscopes, fluorescent probes and labelling chemistry will further refine the resolution of these methods, considerably narrowing the gap between light and electron microscopy. Using a variety of technical strategies, these new technologies have begun to provide insights into cell biology and virology. In particular, real-time imaging methods that track individual virus particles in living cells are being used to study virus assembly, and super-resolution microscopy has already defined interactions with cell factors during HIV-1 budding [55, 56]. In the near future, these microscopies will have an increasing impact in the field of virus morphogenesis.
14.5.2 Correlative Microscopy: From Live Cells to High Resolution
Electron microscopy has contributed more than any other method to our understanding of virus assembly in the cell, although its static nature nonetheless makes it difficult to characterise highly dynamic processes. Correlative light and electron microscopy (CLEM) combines the advantages of live cell imaging with the high resolution of EM. A number of procedures have been reported, and the method of choice depends on the research question . Basically, with CLEM we can select individual live cells with interesting features for a detailed, high-resolution study in TEM and ET. Finding adequate probes for CLEM will be the main technical challenge of these studies.
14.5.3 Clonable Tags for Electron Microscopy and Tomography
Genetically clonable tags for TEM and ET would supply new strategies for the ultrastructural characterisation of virus assembly. To date, two types of approaches have been reported, using photoconversion of fluorescent tags or metal-binding proteins. Diaminobenzidine (DAB) can be photoconverted by production of singlet oxygen from fluorescing proteins to generate dense osmiophilic precipitates that are visible in the electron microscope . The resulting signals are diffuse and lack the resolution of particulate probes, although reasonable results have been obtained for proteins concentrated in cell organelles and in electron tomography [37, 42].
Metal-binding proteins were recently validated as clonable tags for EM in bacteria [58, 59] and mammalian cells . These methods allow detection of intracellular proteins at high sensitivity and molecular scale resolution. To track protein molecules in CLEM, proteins can be double-tagged with a metal-binding peptide and a fluorescent probe. This will give us a new way to look inside cells and visualise where and how individual macromolecules come together to build viral particles.
New technologies have recently begun to offer access to analysis of viruses in cells in unprecedented detail. The complexity of the interaction networks established in these contexts is changing our concept of viruses from that of inert molecular organisms to “live” entities able to carry out a wide variety of activities inside cells.
Also especially recommended for further reading are references [1, 2, 3, 5, 19, 37, 49, 54] listed above.
Our gratitude to Drs German Andrés, Abraham Minsky, Ralf Bartenschlager, Petr Chlanda, Jacomine Krijnse-Locker, Laura Sanz, José Jesús Fernández, Juan Fontana, and Noelia López Montero for providing images, and to Catherine Mark for editorial assistance. Work in C.R.’s laboratory was funded by a grant from the Ministry of Science and Innovation of Spain (BIO2009-07255). I.F.C. is recipient of a fellowship from the FPI Program of the Spanish Ministry of Economy and Competitiveness.
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