Dataset Information

Investigating Tick-borne Flaviviral-like Particles as a Delivery System for Gene Therapy.

ABSTRACT:

Background

Research on the biogenesis of tick-borne encephalitis virus (TBEV) would benefit gene therapy. Due to specific arrangements of genes along the TBEV genome, its viral-like particles (VLPs) could be exploited as shuttles to deliver their replicon, which carries therapeutic genes, to immune system cells.

Objective

To develop a flaviviral vector for gene delivery as a part of gene therapy research that can be expressed in secretable VLP suicidal shuttles and provide abundant unique molecular and structural data supporting this gene therapy concept.

Method

TBEV structural gene constructs of a Swedish Torö strain were cloned into plasmids driven by the promoters CAG and CMV and then transfected into various cell lines, including COS-1 and BHK-21. Time-course sampling of the cells, culture fluid, cell lysate supernatant, and pellet specimens were performed. Western blotting and electron microscopy analyses of collected specimens were used to investigate molecular and structural processing of TBEV structural proteins.

Results

Western blotting analysis showed differences between promoters in directing the gene expression of the VLPs constructs. The premature flaviviral polypeptides as well as mature VLPs could be traced. Using electron microscopy, the premature and mature VLP accumulation in cellular compartments-and also endoplasmic reticulum proliferation as a virus factory platform-were observed in addition to secreted VLPs.

Conclusions

The abundant virologic and cellular findings in this study show the natural processing and safety of inserting flaviviral structural genes into suicidal VLP shuttles. Thus, we propose that these VLPs are a suitable gene delivering system model in gene therapy.

SUBMITTER: Neddermeyer AH

PROVIDER: S-EPMC6076373 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

Investigating Tick-borne Flaviviral-like Particles as a Delivery System for Gene Therapy.

Neddermeyer Anne H AH Hultenby Kjell K Paidikondala Maruthibabu M Schuchman Ryan M RM Bidokhti Mehdi R M MRM

Current therapeutic research, clinical and experimental 20171016

<h4>Background</h4>Research on the biogenesis of tick-borne encephalitis virus (TBEV) would benefit gene therapy. Due to specific arrangements of genes along the TBEV genome, its viral-like particles (VLPs) could be exploited as shuttles to deliver their replicon, which carries therapeutic genes, to immune system cells.<h4>Objective</h4>To develop a flaviviral vector for gene delivery as a part of gene therapy research that can be expressed in secretable VLP suicidal shuttles and provide abundan ...[more]

PMID: 30093925

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Project description:Flaviviruses assemble in the endoplasmic reticulum by a mechanism that appears to be driven by lateral interactions between heterodimers of the envelope glycoproteins E and prM. Immature intracellular virus particles are then transported through the secretory pathway and converted to their mature form by cleavage of the prM protein by the cellular protease furin. Earlier studies showed that when the prM and E proteins of tick-borne encephalitis virus are expressed together in mammalian cells, they assemble into membrane-containing, icosahedrally symmetrical recombinant subviral particles (RSPs), which are smaller than whole virions but retain functional properties and undergo cleavage maturation, yielding a mature form in which the E proteins are arranged in a regular T = 1 icosahedral lattice. In this study, we generated immature subviral particles by mutation of the furin recognition site in prM. The mutation resulted in the secretion of two distinct size classes of particles that could be separated by sucrose gradient centrifugation. Electron microscopy showed that the smaller particles were approximately the same size as the previously described mature RSPs, whereas the larger particles were approximately the same size as the virus. Particles of the larger size class were also detected with a wild-type construct that allowed prM cleavage, although in this case the smaller size class was far more prevalent. Subtle differences in endoglycosidase sensitivity patterns suggested that, in contrast to the small particles, the E glycoproteins in the large subviral particles and whole virions might be in nonequivalent structural environments during intracellular transport, with a portion of them inaccessible to cellular glycan processing enzymes. These proteins thus appear to have the intrinsic ability to form alternative assembly products that could provide important clues about the role of lateral envelope protein interactions in flavivirus assembly.

Dataset Information

Investigating Tick-borne Flaviviral-like Particles as a Delivery System for Gene Therapy.

Background

Objective

Method

Results

Conclusions

Publications

Investigating Tick-borne Flaviviral-like Particles as a Delivery System for Gene Therapy.

Similar Datasets

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