scholarly journals Enhanced Local Symmetry Interactions Globally Stabilize a Mutant Virus Capsid That Maintains Infectivity and Capsid Dynamics

2006 ◽  
Vol 80 (7) ◽  
pp. 3582-3591 ◽  
Author(s):  
Jeffrey A. Speir ◽  
Brian Bothner ◽  
Chunxu Qu ◽  
Deborah A. Willits ◽  
Mark J. Young ◽  
...  
Keyword(s):  
Critical Role ◽  
Local Symmetry ◽  
High Salt ◽  
Direct Result ◽  
Wild Type ◽  
Particle Structure ◽  
Cowpea Chlorotic Mottle Virus ◽  
Chlorotic Mottle Virus ◽  
Chlorotic Mottle

ABSTRACT Structural transitions in viral capsids play a critical role in the virus life cycle, including assembly, disassembly, and release of the packaged nucleic acid. Cowpea chlorotic mottle virus (CCMV) undergoes a well-studied reversible structural expansion in vitro in which the capsid expands by 10%. The swollen form of the particle can be completely disassembled by increasing the salt concentration to 1 M. Remarkably, a single-residue mutant of the CCMV N-terminal arm, K42R, is not susceptible to dissociation in high salt (salt-stable CCMV [SS-CCMV]) and retains 70% of wild-type infectivity. We present the combined structural and biophysical basis for the chemical stability and viability of the SS-CCMV particles. A 2.7-Å resolution crystal structure of the SS-CCMV capsid shows an addition of 660 new intersubunit interactions per particle at the center of the 20 hexameric capsomeres, which are a direct result of the K42R mutation. Protease-based mapping experiments of intact particles demonstrate that both the swollen and closed forms of the wild-type and SS-CCMV particles have highly dynamic N-terminal regions, yet the SS-CCMV particles are more resistant to degradation. Thus, the increase in SS-CCMV particle stability is a result of concentrated tethering of subunits at a local symmetry interface (i.e., quasi-sixfold axes) that does not interfere with the function of other key symmetry interfaces (i.e., fivefold, twofold, quasi-threefold axes). The result is a particle that is still dynamic but insensitive to high salt due to a new series of bonds that are resistant to high ionic strength and preserve the overall particle structure.

Expression and Self Assembly of Cowpea Chlorotic Mottle Virus Capsid Proteins inPichia pastorisand Encapsulation of Fluorescent Myoglobin

2011 ◽  
Vol 1317 ◽  
Author(s):  
Yuanzheng Wu ◽  
Hetong Yang ◽  
Hyun-Jae Shin
Keyword(s):  
Coat Protein ◽  
Large Scale ◽  
Materials Science ◽  
Cesium Chloride ◽  
Size Exclusion ◽  
Mottle Virus ◽  
Cowpea Chlorotic Mottle Virus ◽  
Chlorotic Mottle Virus ◽  
Chlorotic Mottle

Abstract:Cowpea chlorotic mottle virus (CCMV) has been a model system for virus studies for over 40 years and now is considered to be a perfect candidate as nanoplatform for applications in materials science and medicine. The ability of CCMV to self assemblein vitrointo virus-like particles (VLPs) or capsids makes an ideal reaction vessel for nanomaterial synthesis and entrapment. Here we report expression of codon optimized CCMV coat protein inPichia pastorisand production of self assembled CCMV VLPs by large-scale fermentation. CCMV coat protein gene (573 bp) was synthesized according to codon preference ofP. pastorisand cloned into pPICZA vector. The recombinant plasmid pPICZA-CP was transformed intoP. pastorisGS115 by electroporation. The resulting yeast colonies were screened by PCR and analyzed for protein expression by SDS-PAGE. After large-scale fermentation CCMV coat protein yields reached 4.8 g L−1. The CCMV VLPs were purified by modified PEG precipitation followed by cesium chloride density gradient ultracentrifugation, and then analyzed by size exclusion fast performance liquid chromatography (FPLC), UV spectrometry and transmission electron microscopy. Myoglobin was used as a model protein to be encapsulated in CCMV VLPs. The fluorescence spectroscopy showed that inclusion of myoglobin had occurred. The results indicated the production of CCMV capsids byP. pastorisfermentation now available for utilization in pharmacology or nanotechnology fields.

scholarly journals Structural Transitions and Energy Landscape for Cowpea Chlorotic Mottle Virus Capsid Mechanics from Nanomanipulation in Vitro and in Silico

2013 ◽  
Vol 105 (8) ◽  
pp. 1893-1903 ◽  
Author(s):  
Olga Kononova ◽  
Joost Snijder ◽  
Melanie Brasch ◽  
Jeroen Cornelissen ◽  
Ruxandra I. Dima ◽  
...  
Keyword(s):  
In Silico ◽  
Energy Landscape ◽  
Mottle Virus ◽  
Structural Transitions ◽  
Virus Capsid ◽  
Cowpea Chlorotic Mottle Virus ◽  
Chlorotic Mottle Virus ◽  
Chlorotic Mottle

scholarly journals In Vitro Assembly of Cowpea Chlorotic Mottle Virus from Coat Protein Expressed in Escherichia coli and in Vitro-Transcribed Viral cDNA

Virology ◽  
1995 ◽  
Vol 207 (2) ◽  
pp. 486-494 ◽  
Author(s):  
Xiaoxia Zhao ◽  
James M. Fox ◽  
Norman H. Olson ◽  
Timothy S. Baker ◽  
Mark J. Young
Keyword(s):  
Escherichia Coli ◽  
Coat Protein ◽  
Mottle Virus ◽  
Cowpea Chlorotic Mottle Virus ◽  
Chlorotic Mottle Virus ◽  
Chlorotic Mottle ◽  
In Vitro Assembly ◽  
Viral Cdna

scholarly journals The impact of size on particle drainage dynamics and antibody response

2020 ◽  
Author(s):  
Simon Zinkhan ◽  
Anete Ogrina ◽  
Ina Balke ◽  
Gunta Reseviča ◽  
Andris Zeltins ◽  
...  
Keyword(s):  
Immune Response ◽  
B Cells ◽  
Lymphoid Organs ◽  
Cowpea Chlorotic Mottle Virus ◽  
Chlorotic Mottle Virus ◽  
Chlorotic Mottle ◽  
Secondary Lymphoid Organs ◽  
The Impact

AbstractVaccine-induced immune response can be greatly enhanced by mimicking pathogen properties. The size and the repetitive geometric shape of virus-like particles (VLPs) influence their immunogenicity by facilitating drainage to secondary lymphoid organs and enhancing interaction with and activation of B-cells and other innate humoral immune components. VLPs derived from the plant Bromovirus genus, specifically cowpea chlorotic mottle virus (CCMV), are T=3 icosahedron particles. They can be easily expressed in an E. coli host system and package ssRNA during the expression process. Recently, we have engineered CCMV-VLPs by incorporating the universal tetanus toxoid (TT) epitope at the N-terminus. The modified CCMVTT-VLPs successfully form icosahedral particles T=3, with a diameter of ∼30nm analogous to the parental VLPs. Interestingly, incorporating TT epitope at the C-terminus of CCMVTT-VLPs results in the formation of Rod-shaped VLPs, ∼1µm in length and ∼30nm in width. In this study, we have investigated the draining kinetics and immunogenicity of both engineered forms (termed as Round-shaped CCMVTT-VLPs and Rod-shaped CCMVTT-VLPs) as potential B cell immunogens using different in vitro and in vivo assays. Our results reveal that Round-shaped CCMVTT-VLPs are more efficient in draining to secondary lymphoid organs to charge antigen-presenting cells as well as B-cells. Furthermore, compared to Rod-shaped CCMVTT-VLPs, Round-shaped CCMVTT-VLPs led to more than 100-fold increased systemic IgG and IgA responses accompanied by prominent formation of splenic germinal centers. Round-shaped CCMVTT-VLPs could also polarize the induced immune response towards TH1. Up to our knowledge, this is the first study investigating and comparing the draining kinetics and immunogenicity of one and the same VLP monomer forming nano-sized icosahedrons or rods in the micrometer size.

Assembly of a spherical plant virus

1976 ◽  
Vol 276 (943) ◽  
pp. 113-122 ◽  
Keyword(s):  
Buoyant Density ◽  
Sedimentation Coefficient ◽  
Protein Subunit ◽  
Cowpea Chlorotic Mottle Virus ◽  
Average Mass ◽  
Chlorotic Mottle Virus ◽  
Chlorotic Mottle ◽  
Average Molecular Masses

The conditions previously reported as necessary for the reassembly of spherical viruses have been distinctly unphysiological and such reassembly cannot be related directly to the in vivo reaction. Mild conditions for the in vitro reassembly of cowpea chlorotic mottle virus (CCMV) from its isolated components have now been described (Adolph & Butler 1975) and the reassembled virus characterized. This reassembly involved the co-aggregation of the RNA and protein around neutrality and at ionic strength 0.2, giving yields of 70% encapsidation at pH 6.0. The reaction was independent of temperature over the range 5-25 °C and did not require the presence of Mg 2+ ions. The reassembled virus shows a stability similar to that of native CCMV, with the same change in sedimentation coefficient around pH 6.5. The molecular mass and buoyant density in CsCl are also the same as those of native CCMV, while the electron microscope reveals a surface morphology on the reassembled particles like that on native CCMV. Analysis of the number-average, mass-average, and Z-average molecular masses of the purified protein at both pH 6.0 and pH 7.5 suggests that the active unit for reassembly is a dimer of the protein subunit.

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