Asymmetric analysis reveals novel virus capsid features
- 773 Downloads
Cryo-electron microscopy and single-particle image analysis are frequently used methods for macromolecular structure determination. Conventional single-particle analysis, however, usually takes advantage of inherent sample symmetries which assist in the calculation of the structure of interest (such as viruses). Many viruses assemble an icosahedral capsid and often icosahedral symmetry is applied during structure determination. Symmetry imposition, however, results in the loss of asymmetric features of the virus. Here, we provide a brief overview of the methods used to investigate non-symmetric capsid features. These include the recently developed focussed classification as well as more conventional methods which simply do not impose any symmetry. Asymmetric single-particle image analysis can reveal novel aspects of virus structure. For example, the VP4 capsid spike of rotavirus is only present at partial occupancy, the bacteriophage MS2 capsid contains a single copy of a maturation protein and some viruses also encode portals or portal-like assemblies for the packaging and/or release of their genome upon infection. Advances in single-particle image reconstruction methods now permit novel discoveries from previous single-particle data sets which are expanding our understanding of fundamental aspects of virus biology such as viral entry and egress.
KeywordsCryo-electron microscopy Asymmetry Virus Capsid Portal
Transmission electron microscopy has been widely used for the visualisation of biological macromolecules, including viruses, for several decades. In 1959, the now widely used method of negative staining was first described (Brenner and Horne 1959). Embedding macromolecular assemblies in a layer of heavy-metal salt provides excellent image contrast for sample evaluation, but is not suited to high-resolution structure determination. The demonstration of imaging frozen hydrated specimens at low temperature in the transmission electron microscope (cryoEM) in 1984 (Adrian et al. 1984) led to the emergence of a new field in structural biology. CryoEM permits the faithful imaging of biological molecules in a close-to-native state. Images may then be processed computationally to extract 3D structure information. For much of its history, cryoEM yielded 3D reconstructions at modest resolution and was not able to provide reliable information at the atomic level. Recent technological advances have, however, resulted in a resolution capability that rivals that of X-ray crystallography leading to the calculation of maps with very well-defined features, such as the recently solved structure of human apo-ferritin that was determined at 1.65Å resolution (Adrian et al. 1984; Zivanov et al. 2018). This advance in resolution of structures solved using cryo-electron microscopy (cryoEM) is largely due to developments such as direct detection devices and full automation of data collection in microscopes that may be kept cold for many days at a time. Software advances have likewise been important in extracting maximum information from image data. The field is developing further with the use of cold field emission guns which have already resulted in a 1.54Å structure of apo-ferritin (accession no. EMPIAR-10248), the highest resolution achieved to date using cryoEM (Iudin et al. 2016).
Single-particle analysis (SPA) is a commonly used method for determining the structure of a protein or macromolecular complex by cryoEM. This often involves imposing symmetry on the object of interest i.e. icosahedral symmetry on many virions and viral capsids. This allows the user to take advantage of inherent sample regularities/repeating units to assist in the structure determination process. Whilst this has proven to be a reliable and successful method of 3D reconstruction, it results in the loss of irregular, non-symmetric structural features which may be present in the object.
Probing asymmetry in objects that exhibit inherent symmetry can be challenging if the asymmetric feature is small or presents weak signal. In many cases, performing single-particle reconstruction without imposing symmetry, will nonetheless lead to a perfectly symmetric structure, owing to the dominating effect of strong symmetric features. A notable exception is that of the tailed bacteriophages, where a large tail assembly guarantees correct alignment of the asymmetric object (Tang et al. 2010). Asymmetric or ‘relaxed symmetry’ methods have yielded many informative structures at intermediate resolution in such cases.
The recent adoption of novel asymmetric 3D analysis techniques (of cryoEM SPA data sets) has resulted in a number of significant virological findings concerning smaller asymmetric features including portal-like assemblies and unique capsid proteins. Such studies are critical in expanding our understanding of key events in virus biology, such as morphogenesis and cell entry. Here, we review some recent studies, showing how asymmetric reconstruction methods have been used to identify novel or unexpected features of viruses.
Eccentrically positioned capsids within enveloped virions
Visualising virus-membrane interactions
Picornaviruses are non-enveloped, positive-sense RNA viruses which enter host cells via endocytosis through the formation of ‘intermediate’ particles (Guttman and Baltimore 1977). Picornaviruses exhibit T = 1 and pseudo T = 3 symmetry with the capsids composed of VP1, VP2, VP3 and VP4 proteins which together form the 60 repeating structural units that assemble into the capsid structure (Muckelbauer et al. 1995). A picornavirus, Coxsackievirus B3 (CVB3), has been shown to form an entry intermediate (termed the A-particle) during the early stages of infection (Milstone et al. 2005). Lee et al. used nanodisc technology to mimic the receptor decorated membrane encountered by the virus during infection of host cells and imaged the nanodisc-bound particles using cryoEM followed by both icosahedral and asymmetric reconstruction methods (Lee et al. 2016). A pore was visualised in the CVB3 A-particle in the location adjacent to the nanodisc at a unique three-fold axis, representing the asymmetric features in this region. The weak density/pore in this region was described as being due to the flexible extrusions from capsid proteins VP1, VP2 and VP4. These protein extrusions were only visible at this location adjacent to the nanodisc and could only be visualised after determining the orientation of the particles using icosahedral symmetry followed by the relaxation of this symmetry to generate an asymmetric reconstruction of the CVB3 A-particle (Lee et al. 2016).
Resolving features with partial/low occupancy
Visualising low copy number proteins and viral genomes
Using localised reconstruction to resolve symmetry mismatches
The archaeal virus known as SH1 infects Haloarcula hispanica and has a double-stranded DNA genome which is encapsidated inside an internal membrane that is surrounded by a T = 28 icosahedral shell (Jaalinoja et al. 2008). SH1 virions are approx. 100 nm in diameter and are composed of capsid hexamers, decorated with 2 or 3 turret structures. Additionally, horn-like spikes with 2-fold symmetry are present at the 5-fold symmetry axes of the virion (Jaalinoja et al. 2008). Colibus et al. used a combination of asymmetric reconstruction (no symmetry imposed) followed by the localised reconstruction method described by Ilca et al. (see Fig. 2a) with 2-fold symmetry to resolve the virion and the horn-like spikes on the surface, respectively (see Fig. 3c) (De Colibus et al. 2019, Ilca et al. 2015).
Structure determination of in virio macromolecular complexes
Asymmetric portal assemblies in viruses
Feline calicivirus (FCV) is classified in the caliciviridae (alongside the clinically relevant Norovirus) and encodes a single-stranded, positive-sense RNA genome encapsidated within a T = 3 icosahedral capsid. The capsid is formed of 180 copies of the major capsid protein VP1 which assemble with dimer-clustering, adopting slightly differing conformations, termed A/B and C/C as for bacteriophage MS2 and Qβ. Upon binding its receptor (feline junctional adhesion molecule A), a conformational change occurs within the capsid resulting in a 15° anti-clockwise rotation of the capsomeres (Conley et al. 2019; Bhella and Goodfellow 2011; Bhella et al. 2008). These conformational changes caused blurring of the density in the protruding regions of the A/B and C/C dimers. To overcome this, focussed classification (Mcelwee et al. 2018; Scheres 2016; Zhou et al. 2015) was used to reveal the different conformations of the A/B and C/C capsomeres. In the course of this analysis, a novel portal-like assembly (formed of 12 copies of the VP2 minor capsid protein) was identified at a unique 3-fold symmetry axis (see Fig. 5c, d). The conformational changes observed in VP1 allow for the extrusion of the previously encapsidated VP2 proteins as well as the formation of a small pore in the capsid shell. It is hypothesised that the VP2 portal-like assembly functions as a method of endosomal escape and genome delivery, a process not well understood in positive-sense RNA containing viruses such as the caliciviridae (Conley et al. 2019).
An increasing number of asymmetric 3D analysis methods for cryoEM data sets are emerging which are resulting in the discovery of novel capsid features. Basic asymmetric analysis (imposing no symmetry) has become commonplace in the cryoEM field with other methods such as focussed classification and localised reconstruction being increasingly adopted. These techniques are informing advancements in the field of virology with both known structures being better understood e.g. rotavirus spike occupancy (Ilca et al. 2015) as well as the identification of new structures previously unseen with icosahedral reconstruction, e.g. FCV portal-like assembly (Conley et al. 2019).
Compliance with ethical standards
Conflict of interest
M.J. Conley declares that she has no conflict of interest. D. Bhella declares that he has no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Bhella D, Goodfellow IG (2011) The cryo-electron microscopy structure of feline calicivirus bound to junctional adhesion molecule A at 9-angstrom resolution reveals receptor-induced flexibility and two distinct conformational changes in the capsid protein VP1. J Virol 85:11381–11390CrossRefGoogle Scholar
- Ilca SL, Kotecha A, Sun XY, Poranen MM, Stuart DI, Huiskonen JT (2015) Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes. Nat Commun 6Google Scholar
- Scheres SHW (2016) Processing of structurally heterogeneous cryo-EM data in RELION. Resol Rev: Recent Advances in Cryoem 579:125–157Google Scholar
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.