Microscopy & Microtechniques
Seeing is Believing: High-Resolution Cryo Electron Microscopy reveals Mechanism of Virus Assembly and Genome Encapsidation
Dr Emma Hesketh and Rebecca Thompson
In new work led by researchers at the University of Leeds’ Astbury Centre for Structural Molecular Biology, recent advances in cryo-electron microscopy (cryo-EM) were used to investigate the structure of a small plant virus, Cowpea mosaic virus (CPMV). The work* sought to reveal the mechanism of capsid assembly and genome encapsidation of CPMV, laying the groundwork for use of this virus capsid in biotechnology applications.
CPMV can replicate to produce many billions of identical copies of itself in plants. The virus is comprised of a hollow sphere of protein, or capsid, which is in turn made up of multiple copies of the ‘small’ and ‘large’ subunits. Inside this capsid is the virus’ single stranded RNA genome. The research sought to investigate how the capsid assembles and in particular how it packages its genome.
This study, which was a collaboration with colleagues at the John Innes Centre (JIC) in Norwich, builds on previous, award-winning research, in which Professor George Lomonossoff and colleagues at the JIC developed a system to create ‘empty virus-like particles’ (eVLPs); the CPMV capsid without the infectious RNA inside. As the eVLP has no genome, it cannot reproduce itself or mutate, making it ideal for use in biotechnology. The capsid could be engineered by changing the protein sequence to target different capsids for different purposes. However, to achieve this level of engineering, we need precise, atomic level information about the capsid structure. Eventually CPMV eVLPs could be designed as vehicles for targeted drug delivery, a source of novel diagnostic reagents, or even new vaccines.
This new research led by researchers in Leeds, shows the structure of the eVLP in unprecedented detail. CPMV is one of the smallest known viruses and so its direct visualisation requires use of an electron microscope. Cryo-EM images, such as shown in fi gure 1, were processed to generate structures of both CPMV and the related eVLP to 3–3.5 Ångstroms (Å) shown in fi gures 2 and 3.
Figure 6. Researchers from the University of Leeds. Left to right Emma Hesketh, Neil Ranson, Rebecca Thompson.
Dr Neil Ranson said, “At this resolution, which rivals that achieved by X-ray crystallography, the side-chains of the individual amino acids that form the protein capsid could be built into position, providing an atomic model of the entire structure. This opens the way to manipulating those amino acids and intervening in the function of the protein capsid with unprecedented precision.”
The researchers show how CPMV is a highly symmetrical protein shell formed from fi ve-sided ‘pentons’ each built from fi ve copies of an asymmetric subunit. Each asymmetric unit is made up of the large subunit (green) and the small subunit (blue). In fi gure 4 we can clearly see the single stranded RNA genome of CPMV inside the protein shell. The basic protein subunit is very simple, so the virus only needs a very small amount of genetic information to make a large protein shell. Not only is this very effi cient, but CPMV capsids are highly stable, another characteristic which may be exploited in biotechnological applications.
Figure 1. Cryo electron micrographs of empty CPMV particles (eVLPs, left) and ‘full’ CPMV particles containing its single stranded RNA genome (right).
Figure 2. The structure of an empty CPMV (eVLP) produced using cryo electron microscopy.
Figure 3. The structure of CPMV produced using cryo electron microscopy.
Previously, biochemical studies by the JIC team predicted that a small, 24 residue extension at one end of the small subunits of the capsid was involved in encapsidation of the RNA genome during virus assembly. However, this feature had not been previously observed using other structural techniques such as X-ray crystallography, making it diffi cult to validate its role in assembly. In this work, the cryo-EM structures allowed the researchers to visualise the extension for the fi rst time (see the pink section in fi gure 5). It found that the 24 residue extension of the S subunit holds the pentons together as the virus’ outer structure is built. These residues are also essential for the virus to package its genes, but it is cleaved from the virus when it has done its job.
Figure 5. The structure of the 24 residue extension visualised for the fi rst time in this study.
INTERNATIONAL LABMATE - JANUARY/FEBRUARY 2016
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