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Mechanics of Virus-like Particles Labeled with Green Fluorescent Protein.

ABSTRACT: Nanoindentation with an atomic force microscope was used to investigate the mechanical properties of virus-like particles (VLPs) derived from the avian pathogen infectious bursal disease virus, in which the major capsid protein was modified by fusion with enhanced green fluorescent protein (EGFP). These VLPs assemble as ?70-nm-diameter T = 13 icosahedral capsids with large cargo space. The effect of the insertion of heterologous proteins in the capsid was characterized in the elastic regime, revealing that EGFP-labeled chimeric VLPs are more rigid than unmodified VLPs. In addition, nanoindentation measurements beyond the elastic regime allowed the determination of brittleness and rupture force limit. EGFP incorporation results in a complex shape of the indentation curve and lower critical indentation depth of the capsid, rendering more brittle particles as compared to unlabeled VLPs. These observations suggest the presence of a complex and more constrained network of interactions between EGFP and the capsid inner shell. These results highlight the effect of fluorescent protein insertion on the mechanical properties of these capsids. Because the physical properties of the viral capsid are connected to viral infectivity and VLP transport and disassembly, our results are relevant to design improved labeling strategies for fluorescence tracking in living cells.

SUBMITTER: Mertens J

PROVIDER: S-EPMC6260219 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Mechanics of Virus-like Particles Labeled with Green Fluorescent Protein.

Mertens Johann J Bondia Patricia P Allende-Ballestero Carolina C Carrascosa José L JL Flors Cristina C Castón José R JR

Biophysical journal 20180901 8

Nanoindentation with an atomic force microscope was used to investigate the mechanical properties of virus-like particles (VLPs) derived from the avian pathogen infectious bursal disease virus, in which the major capsid protein was modified by fusion with enhanced green fluorescent protein (EGFP). These VLPs assemble as ∼70-nm-diameter T = 13 icosahedral capsids with large cargo space. The effect of the insertion of heterologous proteins in the capsid was characterized in the elastic regime, rev ...[more]

PMID: 30249401

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Project description:UnlabelledWe generated a recombinant Akabane virus (AKAV) expressing enhanced green fluorescence protein (eGFP-AKAV) by using reverse genetics. We artificially constructed an ambisense AKAV S genome encoding N/NSs on the negative-sense strand, and eGFP on the positive-sense strand with an intergenic region (IGR) derived from the Rift Valley fever virus (RVFV) S genome. The recombinant virus exhibited eGFP fluorescence and had a cytopathic effect in cell cultures, even after several passages. These results indicate that the gene encoding eGFP in the ambisense RNA could be stably maintained. Transcription of N/NSs and eGFP mRNAs of eGFP-AKAV was terminated within the IGR. The mechanism responsible for this appears to be different from that in RVFV, where the termination sites for N and NSs are determined by a defined signal sequence. We inoculated suckling mice intraperitoneally with eGFP-AKAV, which resulted in neurological signs and lethality equivalent to those seen for the parent AKAV. Fluorescence from eGFP in frozen brain slices from the eGFP-AKAV-infected mice was localized to the cerebellum, pons, and medulla oblongata. Our approach to producing a fluorescent virus, using an ambisense genome, helped obtain eGFP-AKAV, a fluorescent bunyavirus whose viral genes are intact and which can be easily visualized.ImportanceAKAV is the etiological agent of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic loss to the livestock industry. We successfully generated a recombinant enhanced green fluorescent protein-tagged AKAV containing an artificial ambisense S genome. This virus could become a useful tool for analyzing AKAV pathogenesis in host animals. In addition, our approach of using an ambisense genome to generate an orthobunyavirus stably expressing a foreign gene could contribute to establishing alternative vaccine strategies, such as bivalent vaccine virus constructs, for veterinary use against infectious diseases.

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Mechanics of Virus-like Particles Labeled with Green Fluorescent Protein.

Publications

Mechanics of Virus-like Particles Labeled with Green Fluorescent Protein.

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