Atomic cryo-EM structures of viruses

Jiang, Wen; Tang, Liang

doi:10.1016/j.sbi.2017.07.002

Cited by 60 publications

(37 citation statements)

References 77 publications

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“…Since viral structures and EVs are known to have an overall negative charge at physiological pH [18,19], this strategy could improve their adsorption and reduce nanoparticle loss during the sample preparation process. Cryo-TEM has also gained increasing interest as a tool for nanoparticle visualization over the last years [13]. Essentially, this technique enables the visualization of viruses and VLPs in their native conformation at nanometric and even atomic scale [3], and the addition of a contrast solution is not required.…”

Section: Introductionmentioning

confidence: 99%

Quality Assessment of Virus-Like Particles at Single Particle Level: A Comparative Study

Puente‐Massaguer

Cervera

Gòdia

2020

Viruses

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Virus-like particles (VLPs) have emerged as a powerful scaffold for antigen presentation and delivery strategies. Compared to single protein-based therapeutics, quality assessment requires a higher degree of refinement due to the structure of VLPs and their similar properties to extracellular vesicles (EVs). Advances in the field of nanotechnology with single particle and high-resolution analysis techniques provide appealing approaches to VLP characterization. In this study, six different biophysical methods have been assessed for the characterization of HIV-1-based VLPs produced in mammalian and insect cell platforms. Sample preparation and equipment set-up were optimized for the six strategies evaluated. Electron Microscopy (EM) disclosed the presence of several types of EVs within VLP preparations and cryogenic transmission electron microscopy (cryo-TEM) resulted in the best technique to resolve the VLP ultrastructure. The use of super-resolution fluorescence microscopy (SRFM), nanoparticle tracking analysis (NTA) and flow virometry enabled the high throughput quantification of VLPs. Interestingly, differences in the determination of nanoparticle concentration were observed between techniques. Moreover, NTA and flow virometry allowed the quantification of both EVs and VLPs within the same experiment while analyzing particle size distribution (PSD), simultaneously. These results provide new insights into the use of different analytical tools to monitor the production of nanoparticle-based biologicals and their associated contaminants.

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Section: Introductionmentioning

confidence: 99%

Quality Assessment of Virus-Like Particles at Single Particle Level: A Comparative Study

Puente‐Massaguer

Cervera

Gòdia

2020

Viruses

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“…Therefore, biophysical methods are needed to ensure the structural integrity of these nanoparticles. Analytical ultracentrifugation and dynamic light scattering (DLS) were primarily used to this end, whereas electron microscopy (EM) has been the preferred methodology for VLP visualization, as nanometric or even atomic resolution can be achieved (Jiang & Tang, 2017). Alternatively, atomic force microscopy (AFM) has emerged as a powerful tool to study viruses (de Pablo, 2018).…”

Section: Introductionmentioning

confidence: 99%

Quantification of the HIV‐1 virus‐like particle production process by super‐resolution imaging: From VLP budding to nanoparticle analysis

Puente‐Massaguer

Cervera

Gòdia

2020

Biotech & Bioengineering

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Virus‐like particles (VLPs) offer great promise in the field of nanomedicine. Enveloped VLPs are a class of these nanoparticles and their production process occurs by a budding process, which is known to be the most critical step at intracellular level. In this study, we developed a novel imaging method based on super‐resolution fluorescence microscopy (SRFM) to assess the generation of VLPs in living cells. This methodology was applied to study the production of Gag VLPs in three animal cell platforms of reference: HEK 293‐transient gene expression (TGE), High Five‐baculovirus expression vector system (BEVS) and Sf9‐BEVS. Quantification of the number of VLP assembly sites per cell ranged from 500 to 3,000 in the different systems evaluated. Although the BEVS was superior in terms of Gag polyprotein expression, the HEK 293‐TGE platform was more efficient regarding the assembly of Gag as VLPs. This was translated into higher levels of non‐assembled Gag monomer in BEVS harvested supernatants. Furthermore, the presence of contaminating nanoparticles was evidenced in all three systems, specifically in High Five cells. The SRFM‐based method here developed was also successfully applied to measure the concentration of VLPs in crude supernatants. The lipid membrane of VLPs and the presence of nucleic acids alongside these nanoparticles could also be detected using common staining procedures. Overall, a complete picture of the VLP production process was achieved in these three production platforms. The robustness and sensitivity of this new approach broaden the applicability of SRFM toward the development of new detection, diagnosis and quantification methods based on confocal microscopy in living systems.

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“…Arguably the most common example of symmetry mismatches in icosahedral viruses is the portal protein, which was originally discovered in tailed bacteriophage, although symmetry mismatches are present in viruses that infect all kingdoms of life. Spherical capsids with T = 7 symmetry have been enormously well studied in the last two decades, thanks to increased access to better technology within the field of cryo-electron microscopy (cryo-EM), which is ideally suited to image complex molecular machines [ 22 , 23 , 24 , 25 , 26 ]. A wealth of information regarding larger and more complex capsid geometries has also benefitted from current advances in environmental isolation methodology (“phage hunting”) [ 27 , 28 , 29 ] and access to better laboratory culture schemes, revealing an unprecedented breadth of capsid geometry.…”

Section: Principles Of Icosahedral Virus Assemblymentioning

confidence: 99%

Breaking Symmetry in Viral Icosahedral Capsids as Seen through the Lenses of X-ray Crystallography and Cryo-Electron Microscopy

Parent

Schrad

Cingolani

2018

Viruses

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The majority of viruses on Earth form capsids built by multiple copies of one or more types of a coat protein arranged with 532 symmetry, generating an icosahedral shell. This highly repetitive structure is ideal to closely pack identical protein subunits and to enclose the nucleic acid genomes. However, the icosahedral capsid is not merely a passive cage but undergoes dynamic events to promote packaging, maturation and the transfer of the viral genome into the host. These essential processes are often mediated by proteinaceous complexes that interrupt the shell’s icosahedral symmetry, providing a gateway through the capsid. In this review, we take an inventory of molecular structures observed either internally, or at the 5-fold vertices of icosahedral DNA viruses that infect bacteria, archea and eukaryotes. Taking advantage of the recent revolution in cryo-electron microscopy (cryo-EM) and building upon a wealth of crystallographic structures of individual components, we review the design principles of non-icosahedral structural components that interrupt icosahedral symmetry and discuss how these macromolecules play vital roles in genome packaging, ejection and host receptor-binding.

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Atomic cryo-EM structures of viruses

Cited by 60 publications

References 77 publications

Quality Assessment of Virus-Like Particles at Single Particle Level: A Comparative Study

Quality Assessment of Virus-Like Particles at Single Particle Level: A Comparative Study

Quantification of the HIV‐1 virus‐like particle production process by super‐resolution imaging: From VLP budding to nanoparticle analysis

Breaking Symmetry in Viral Icosahedral Capsids as Seen through the Lenses of X-ray Crystallography and Cryo-Electron Microscopy

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