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Structural components for amplification of positive and negative strand VEEV splitzicons

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Title: Structural components for amplification of positive and negative strand VEEV splitzicons
Authors: Blakney, A
McKay, PF
Shattock, R
Item 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.
Issue Date: 26-Jul-2018
Date of Acceptance: 9-Jul-2018
URI: http://hdl.handle.net/10044/1/62348
DOI: https://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/Funder: Engineering & Physical Science Research Council (EPSRC)
Funder's Grant Number: EP/R013764/1
Keywords: RNA
VEEV
amplification
non-structural proteins
replicon
Publication Status: Published
Article Number: ARTN 71
Online Publication Date: 2018-07-09
Appears in Collections:Department of Medicine
Faculty of Medicine



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