Analytical Techniques to Characterize the Structure, Properties, and Assembly of Virus Capsids

Kondylis, Panagiotis; Schlicksup, Christopher John; Zlotnick, Adam; Jacobson, Stephen C.

doi:10.1021/acs.analchem.8b04824

Cited by 36 publications

(39 citation statements)

References 175 publications

(339 reference statements)

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“…This technique distinguishes and quantifies the different species (empty and full particles) by either mass or density without the need for standard material for quantification comparison. Detection and quantification of biotherapeutic particles can also be performed by multi-angle light scattering (MALS) detectors coupled with size exclusion HPLC or asymmetrical field flow fractionation [163,164]. This quantification method does not require a calibration sample since the particle concentration can be directly deduced from the scattered light.…”

Section: Analytics In Process Developmentmentioning

confidence: 99%

Current challenges in biotherapeutic particles manufacturing

Moleirinho

Silva

Alves

et al. 2019

Expert Opinion on Biological Therapy

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Introduction: The development of novel complex biotherapeutics led to new challenges in biopharmaceutical industry. The potential of these particles has been demonstrated by the approval of several products, in the different fields of gene therapy, oncolytic therapy, and tumor vaccines. However, their manufacturing still presents challenges related to the high dosages and purity required. Areas covered: The main challenges that biopharmaceutical industry faces today and the most recent developments in the manufacturing of different biotherapeutic particles are reported here. Several unit operations and downstream trains to purify virus, virus-like particles and extracellular vesicles are described. Innovations on the different purification steps are also highlighted with an eye on the implementation of continuous and integrated processes. Expert opinion: Manufacturing platforms that consist of a low number of unit operations, with higheryielding processes and reduced costs will be highly appreciated by the industry. The pipeline of complex therapeutic particles is expanding and there is a clear need for advanced tools and manufacturing capacity. The use of single-use technologies, as well as continuous integrated operations, are gaining ground in the biopharmaceutical industry and should be supported by more accurate and faster analytical methods.

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Section: Analytics In Process Developmentmentioning

confidence: 99%

Current challenges in biotherapeutic particles manufacturing

Moleirinho

Silva

Alves

et al. 2019

Expert Opinion on Biological Therapy

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“…S5 in the supplemental material). CLP formation was detected by dynamic light scattering (DLS), a relatively high-throughput method previously used to quantitate in vitro CLP assembly (42,45), and by negative-stain electron microscopy. DLS measures the translational diffusion of molecules in solution due to Brownian motion (46).…”

Section: Downloaded Frommentioning

confidence: 99%

Berberine Chloride is an Alphavirus Inhibitor That Targets Nucleocapsid Assembly

Wan

Brown

Kielian

2020

mBio

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Alphaviruses are enveloped positive-sense RNA viruses that can cause serious human illnesses such as polyarthritis and encephalitis. Despite their widespread distribution and medical importance, there are no licensed vaccines or antivirals to combat alphavirus infections. Berberine chloride (BBC) is a pan-alphavirus inhibitor that was previously identified in a replicon-based small-molecule screen. This work showed that BBC inhibits alphavirus replication but also suggested that BBC might have additional effects later in the viral life cycle. Here, we show that BBC has late effects that target the virus nucleocapsid (NC) core. Infected cells treated with BBC late in infection were unable to form stable cytoplasmic NCs or assembly intermediates, as assayed by gradient sedimentation. In vitro studies with recombinant capsid protein (Cp) and purified genomic RNA (gRNA) showed that BBC perturbs core-like particle formation and potentially traps the assembly process in intermediate states. Particles produced from BBC-treated cells were less infectious, despite efficient particle production and only minor decreases in genome packaging. In addition, BBC treatment of free virus particles strongly decreased alphavirus infectivity. In contrast, the infectivity of the negative-sense RNA virus vesicular stomatitis virus was resistant to BBC treatment of infected cells or free virus. Together, our data indicate that BBC alters alphavirus Cp-gRNA interactions and oligomerization and suggest that this may cause defects in NC assembly and in disassembly during subsequent virus entry. Thus, BBC may be considered a novel alphavirus NC assembly inhibitor. IMPORTANCE The alphavirus chikungunya virus (CHIKV) is an example of an emerging human pathogen with increased and rapid global spread. Although an acute CHIKV infection is rarely fatal, many patients suffer from debilitating chronic arthralgia for years. Antivirals against chikungunya and other alphaviruses have been identified in vitro, but to date none have been shown to be efficacious and have been licensed for human use. Here, we investigated a small molecule, berberine chloride (BBC), and showed that it inhibited infectious virus production by several alphaviruses including CHIKV. BBC acted on a late step in the alphavirus exit pathway, namely the formation of the nucleocapsid containing the infectious viral RNA. Better understanding of nucleocapsid formation and its inhibition by BBC will provide important information on the mechanisms of infectious alphavirus production and may enable their future targeting in antiviral strategies.

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“…To validate the presence of holey capsids and not the selective dissociation of hexamer-enriched capsids, we analyzed puri ed holey capsids by resistive pulsive sensing (RPS) 46,60 . In RPS, solute displaces a proportional amount of electrolyte from a nanopore, resulting in a de ection of the current that is proportional to the volume of a single particle ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Asymmetrizing an icosahedral virus capsid by hierarchical assembly of subunits with designed asymmetry

Zhao

Wang

Zhang

et al. 2020

Preprint

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Symmetrical protein complexes are ubiquitous in natural biological systems. Many have been reengineered in vitro for chemical and medical applications. Symmetrical viral capsids and their assembly are frequent platforms for these investigations. Lacking a means to create asymmetric capsids may limit broader applications. Here, starting with the homodimeric Hepatitis B Virus capsid protein, we developed a heterodimer, designed a hierarchical assembly pathway, and produced asymmetric capsids. We showed that the heterodimers assemble into hexamers, and such preformed hexamers can nucleate co-assembly, leading to “Janus” capsids with two discrete patches. We removed the hexamer patches specifically and observed asymmetric holey capsids by cryo-EM reconstruction. The resulting holes can be refilled with new engineered dimers. This programmed assembly pathway provides windows for specific engineering and modification inside and outside of the capsid. This strategy can also be generalized to other capsid assembly systems.

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Analytical Techniques to Characterize the Structure, Properties, and Assembly of Virus Capsids

Abstract: Graphical Abstract

Cited by 36 publications

References 175 publications

Current challenges in biotherapeutic particles manufacturing

Current challenges in biotherapeutic particles manufacturing

Berberine Chloride is an Alphavirus Inhibitor That Targets Nucleocapsid Assembly

Asymmetrizing an icosahedral virus capsid by hierarchical assembly of subunits with designed asymmetry

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