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  4. Structural components for amplification of positive and negative strand VEEV splitzicons
 
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Structural components for amplification of positive and negative strand VEEV splitzicons
File(s)
fmolb-05-00071.pdf (4.92 MB)
Published version
Author(s)
Blakney, Anna
McKay, PF
Shattock, Robin
Type
Journal Article
Abstract
RNA is a promising nucleic acid technology for both vaccines and therapeutics, and replicon RNA has gained traction as a next-generation RNA modality. Replicon RNA self-amplifies using a replicase complex derived from alphaviral non-structural proteins and yields higher protein expression than a similar dose of messenger RNA. Here, we debut RNA splitzicons; a split replicon system wherein the non-structural proteins (NSPs) and the gene of interest are encoded on separate RNA molecules, but still exhibit the self-amplification properties of replicon RNA. We designed both positive and negative strand splitzicons encoding firefly luciferase as a reporter protein to determine which structural components, including the 5′ untranslated region (UTR), a 51-nucleotide conserved sequence element (CSE) from the first nonstructural protein, the subgenomic promoter (SGP) and corresponding untranslated region, and an internal ribosomal entry site (IRES) affect amplification. When paired with a NSP construct derived from the whole, wild type replicon, both the positive and negative strand splitzicons were amplified. The combination of the 51nt CSE, subgenomic promoter and untranslated region were imperative for the positive strand splitzicon, while the negative strand was amplified simply with inclusion of the subgenomic promoter. The splitzicons were amplified by NSPs in multiple cell types and show increasing protein expression with increasing doses of NSP. Furthermore, both the positive and negative strand splitzicons continued to amplify over the course of 72 h, up to >100,000-fold. This work demonstrates a system for screening the components required for amplification from the positive and negative strand intermediates of RNA replicons and presents a new approach to RNA replicon technology.
Date Issued
2018-07-26
Date Acceptance
2018-07-09
Citation
Frontiers in Molecular Biosciences, 2018, 5
URI
http://hdl.handle.net/10044/1/62348
DOI
https://www.dx.doi.org/10.3389/fmolb.2018.00071
ISSN
2296-889X
Publisher
Frontiers Media
Journal / Book Title
Frontiers in Molecular Biosciences
Volume
5
Copyright Statement
© 2018 Blakney, McKay and Shattock. This is an open-access article
distributed under the terms of the Creative Commons Attribution License (CC BY).
The use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice.
No use, distribution or reproduction is permitted which does not comply with these
terms.
Sponsor
Engineering & Physical Science Research Council (EPSRC)
Grant Number
EP/R013764/1
Subjects
RNA
VEEV
amplification
non-structural proteins
replicon
Publication Status
Published
Article Number
ARTN 71
Date Publish Online
2018-07-09
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