Assembly of fluorescent chimeric virus-like particles of canine parvovirus in insect cells

Gilbert, Leona; Toivola, Jouni; Lehtomäki, Elina; Donaldson, Logan W.; Käpylä, P; Vuento, Matti; Oker‐Blom, Christian

doi:10.1016/j.bbrc.2003.11.176

Cited by 28 publications

(31 citation statements)

References 51 publications

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“…7A–C). The diffusion coefficient for EGFP (Table 1) was in agreement with those reported previously [19,27,28]. Further, the diffusion times were clearly lower than the diffusion time of the fluorescent EGFP-VP2-14 protein (0.3 ms; Fig.…”

Section: Resultssupporting

confidence: 91%

“…3) and the break down products in the western blots (Fig. 2) are most likely due to proteolysis of the EGFP-VP2 protein constructs as seen also in previous studies [18,19]. From the negatively stained EM micrographs of the VP2 proteins it was obvious, that VP2, VP2-14, VP2-23, VP2-40 as well as the fusion constructs EGFP-VP2, EGFP-VP2-23 and EGFP-VP2-40 were able to form structures resembling VLPs (Fig.…”

Section: Resultssupporting

confidence: 79%

“…4). All particles appeared to have a diameter of approximately 26 nm, displaying a typical VP2 or wild-type CPV structure [18,19]. However, the EGFP-VP2-14 construct appeared not to assemble into similar structures (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…structure/function relationships at the molecular/cellular levels have been carried out. These include deletions of [33] and epitope insertions in the loops of the virion [34], N-terminal fusion of foreign antigens to VP1 [35,36], N-terminal insertion and fusion [17,19,37] or deletions of VP2 [33,38], and C-terminal fusions to VP2 [34,37,39]. …”

Section: Discussionmentioning

confidence: 99%

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Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles

et al. 2006

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Fluorescence correlation spectroscopy (FCS) monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nanoparticles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids) were fused to the C-terminus of the enhanced green fluorescent protein (EGFP). The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs). The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles.

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Section: Resultssupporting

confidence: 91%

Section: Resultssupporting

confidence: 79%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles

et al. 2006

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“…A wide range of trackable VLPs has been generated by labeling one or more viral components with a fluorescent protein (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). Many VLPs have been labeled after particle production/ purification, usually by chemical coupling to (in)organic Figure 2.…”

Section: Discussionmentioning

confidence: 99%

Generation and characterization of a trackable plant-made influenza H5 virus-like particle (VLP) containing enhanced green fluorescent protein (eGFP)

Young¹,

Arthus-Cartier²,

Yam³

et al. 2015

The FASEB Journal

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Medicago, Inc. has developed an efficient virus-like particle (VLP) vaccine production platform using the Nicotiana benthamiana expression system, and currently has influenza-based products targeting seasonal/pandemic hemagglutinin (HA) proteins in advanced clinical trials. We wished to generate a trackable HA-based VLP that would allow us to study both particle assembly in plants and VLP interactions within the mammalian immune system. To this end, a fusion protein was designed, composed of H5 (from influenza A/Indonesia/05/2005 [H5N1]) with enhanced green fluorescent protein (eGFP). Expression of H5-eGFP in N. benthamiana produced brightly fluorescent ∼160 nm particles resembling H5-VLPs. H5-eGFP-VLPs elicited anti-H5 serologic responses in mice comparable to those elicited by H5-VLPs in almost all assays tested (hemagglutination inhibition/IgG(total)/IgG1/IgG2b/IgG2a:IgG1 ratio), as well as a superior anti-GFP IgG response (mean optical density = 2.52 ± 0.16 sem) to that elicited by soluble GFP (mean optical density = 0.12 ± 0.06 sem). Confocal imaging of N. benthamiana cells expressing H5-eGFP displayed large fluorescent accumulations at the cell periphery, and draining lymph nodes from mice given H5-eGFP-VLPs via footpad injection demonstrated bright fluorescence shortly after administration (10 min), providing proof of concept that the H5-eGFP-protein/VLPs could be used to monitor both VLP assembly and immune trafficking. Given these findings, this novel fluorescent reagent will be a powerful tool to gain further fundamental insight into the biology of influenza VLP vaccines.

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Different applications of virus‐like particles in biology and medicine: Vaccination and delivery systems

2015

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Virus-like particles (VLPs) mimic the whole construct of virus particles devoid of viral genome as used in subunit vaccine design. VLPs can elicit efficient protective immunity as direct immunogens compared to soluble antigens co-administered with adjuvants in several booster injections. Up to now, several prokaryotic and eukaryotic systems such as insect, yeast, plant, and E. coli were used to express recombinant proteins, especially for VLP production. Recent studies are also generating VLPs in plants using different transient expression vectors for edible vaccines. VLPs and viral particles have been applied for different functions such as gene therapy, vaccination, nanotechnology, and diagnostics. Herein, we describe VLP production in different systems as well as its applications in biology and medicine.

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Assembly of fluorescent chimeric virus-like particles of canine parvovirus in insect cells

Cited by 28 publications

References 51 publications

Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles

Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles

Generation and characterization of a trackable plant-made influenza H5 virus-like particle (VLP) containing enhanced green fluorescent protein (eGFP)

Different applications of virus‐like particles in biology and medicine: Vaccination and delivery systems

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