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      Assembly/disassembly of a complex icosahedral virus to incorporate heterologous nucleic acids

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          Abstract

          Hollow protein containers are widespread in nature, and include virus capsids as well as eukaryotic and bacterial complexes. Protein cages are studied extensively for applications in nanotechnology, nanomedicine and materials science. Their inner and outer surfaces can be modified chemically or genetically, and the internal cavity can be used to template, store and/or arrange molecular cargos. Virus capsids and virus-like particles (VLP, noninfectious particles) provide versatile platforms for nanoscale bioengineering. Study of capsid protein self-assembly into monodispersed particles, and of VLP structure and biophysics is necessary not only to understand natural processes, but also to infer how these platforms can be redesigned to furnish novel functional VLP. Here we address the assembly dynamics of infectious bursal disease virus (IBDV), a complex icosahedral virus. IBDV has a ~70 nm-diameter T  =  13 capsid with VP2 trimers as the only structural subunits. During capsid assembly, VP2 is synthesized as a precursor (pVP2) whose C terminus is cleaved. The pVP2 C terminus has an amphipathic helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, necessary for control of assembly, 466/456-residue pVP2 intermediates bearing this helix assemble into VLP only when expressed with an N-terminal His 6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for genetic insertion of proteins (cargo space ~78 000 nm 3). We established an in vitro assembly/disassembly system of HT-VP2-466-based VLP for heterologous nucleic acid packaging and/or encapsulation of drugs and other molecules. HT-VP2-466 (empty) capsids were disassembled and reassembled by dialysis against low-salt/basic pH and high-salt/acid pH buffers, respectively, thus illustrating the reversibility in vitro of IBDV capsid assembly. HT-VP2-466 VLP also packed heterologous DNA by non-specific confinement during assembly. These and previous results establish the bases for biotechnological applications based on the IBDV capsid and its ability to incorporate exogenous proteins and nucleic acids.

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          Mechanisms of Virus Assembly

          Viruses are nanoscale entities containing a nucleic acid genome encased in a protein shell called a capsid and in some cases are surrounded by a lipid bilayer membrane. This review summarizes the physics that govern the processes by which capsids assemble within their host cells and in vitro. We describe the thermodynamics and kinetics for the assembly of protein subunits into icosahedral capsid shells and how these are modified in cases in which the capsid assembles around a nucleic acid or on a lipid bilayer. We present experimental and theoretical techniques used to characterize capsid assembly, and we highlight aspects of virus assembly that are likely to receive significant attention in the near future.
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            Applications of viral nanoparticles in medicine.

            Several nanoparticle platforms are currently being developed for applications in medicine, including both synthetic materials and naturally occurring bionanomaterials such as viral nanoparticles (VNPs) and their genome-free counterparts, virus-like particles (VLPs). A broad range of genetic and chemical engineering methods have been established that allow VNP/VLP formulations to carry large payloads of imaging reagents or drugs. Furthermore, targeted VNPs and VLPs can be generated by including peptide ligands on the particle surface. In this article, we highlight state-of-the-art virus engineering principles and discuss recent advances that bring potential biomedical applications a step closer. Viral nanotechnology has now come of age and it will not be long before these formulations assume a prominent role in the clinic. Copyright © 2011 Elsevier Ltd. All rights reserved.
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              Protein delivery using engineered virus-like particles.

              Over the years, researchers have developed several methods to deliver macromolecules into the cytosol and nucleus of living cells. However, there are limitations to all of these methods. The problems include (i) inefficient uptake, (ii) endosomal entrapment, (iii) delivery that is restricted to certain cell types, and (iv) damage to cells in the delivery process. Retroviral vectors are often used for gene delivery; however, integration of the genome of retroviral vector into the host genome can have serious consequences. Here we describe a safe alternative in which virus-like particles (VLPs), derived from an avian retrovirus, are used to deliver protein to cells. We show that these VLPs are a highly adaptable platform that can be used to deliver proteins either as part of Gag fusion proteins (intracellular delivery) or on the surface of VLPs. We generated VLPs that contain Gag-Cre recombinase, Gag-Fcy::Fur, and Gag-human caspase-8 as a proof-of-concept and demonstrated that the encapsidated proteins are active in recipient cells. In addition, we show that murine IFN-γ and human TNF-related apoptosis-inducing ligand can be displayed on the surface of VLPs, and that these modified VLPs can cause the appropriate response in cells, as evidenced by phosphorylation of STAT1 and induction of cell death, respectively.
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                Author and article information

                Journal
                J Phys Condens Matter
                J Phys Condens Matter
                cm
                JCOMEL
                Journal of Physics
                IOP Publishing
                0953-8984
                1361-648X
                13 December 2017
                13 November 2017
                : 29
                : 49
                : 494001
                Affiliations
                Department of Structure of Macromolecules, Centro Nacional de Biotecnología/CSIC , Cantoblanco, Madrid, Spain jrcaston@ 123456cnb.csic.es
                Author notes
                [1]

                Present address: Departamento de Biotecnología, Facultad de Ciencias Experimentales, Universidad Francisco de Vitoria, 28223, Pozuelo de Alarcón, Madrid, Spain.

                [2]

                Dept. Estructura de Macromoléculas, Centro Nacional Biotecnología/CSIC, Campus de Cantoblanco, Darwin 3, 28049 Madrid, Spain.

                Author information
                https://orcid.org/0000-0003-2350-9048
                Article
                cmaa96ec aa96ec JPCM-109858.R1
                10.1088/1361-648X/aa96ec
                7103166
                29083994
                f15dd5da-01ba-43e2-a729-c70aa8f33eda
                © 2017 IOP Publishing Ltd

                This article is made available via the PMC Open Access Subset for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

                History
                : 2 August 2017
                : 17 October 2017
                : 30 October 2017
                Page count
                Pages: 8
                Funding
                Funded by: Spanish Ministry of Economy and Competitivity
                Award ID: BFU2014-54181
                Award ID: BFU2014-55475-R
                Funded by: Comunidad Autónoma de Madrid
                Award ID: S2013/MIT-2807
                Award ID: S2013/MIT-2850
                Categories
                Paper
                Special Issue on Viral Capsids
                Custom metadata
                1361-648X/17/494001+08$33.00
                Printed in the UK
                yes

                virus capsid,assembly/disassembly,virus-like particle,nucleic acid encapsulation,infectious burial disease virus (ibdv)

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