Structure–function analysis of the equine hepacivirus 5′ untranslated region highlights the conservation of translational mechanisms across the hepaciviruses

Lattimer, Joseph, Stewart, Hazel, Locker, Nicolas, Tuplin, Andrew, Stonehouse, Nicola J. and Harris, Mark (2019) Structure–function analysis of the equine hepacivirus 5′ untranslated region highlights the conservation of translational mechanisms across the hepaciviruses Journal of General Virology.

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Abstract

Equine hepacivirus (EHcV) (now also classified as hepacivirus A) is the closest genetic relative to hepatitis C virus (HCV) and is proposed to have diverged from HCV within the last 1000 years. The 5′ untranslated regions (UTRs) of both HCV and EHcV exhibit internal ribosome entry site (IRES) activity, allowing cap-independent translational initiation, yet only the HCV 5′UTR has been systematically analysed. Here, we report a detailed structural and functional analysis of the EHcV 5′UTR. The secondary structure was determined using selective 2′ hydroxyl acylation analysed by primer extension (SHAPE), revealing four stem–loops, termed SLI, SLIA, SLII and SLIII, by analogy to HCV. This guided a mutational analysis of the EHcV 5′UTR, allowing us to investigate the roles of the stem–loops in IRES function. This approach revealed that SLI was not required for EHcV IRES-mediated translation. Conversely, SLIII was essential, specifically SLIIIb, SLIIId and a GGG motif that is conserved across the Hepaciviridae. Further SHAPE analysis provided evidence that this GGG motif mediated interaction with the 40S ribosomal subunit, whilst a CUU sequence in the apical loop of SLIIIb mediated an interaction with eIF3. In addition, we showed that a microRNA122 target sequence located between SLIA and SLII mediated an enhancement of translation in the context of a subgenomic replicon. Taken together, these results highlight the conservation of hepaciviral translation mechanisms, despite divergent primary sequences.

Item Type:	Article
Divisions :	Faculty of Health and Medical Sciences > School of Biosciences and Medicine
Authors :	Name Email ORCID Lattimer, Joseph Stewart, Hazel Locker, Nicolas N.Locker@surrey.ac.uk Tuplin, Andrew Stonehouse, Nicola J. Harris, Mark
Date :	6 September 2019
Funders :	Biotechnology and Biological Sciences Research Council (BBSRC), Wellcome Trust
DOI :	10.1099/jgv.0.001316
Copyright Disclaimer :	© 2019 The Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Uncontrolled Keywords :	Equine hepacivirus; 5′ untranslated region; Selective 2′ hydroxyl acylation analysed by primer extension; Internal ribosome entry site; Translation
Depositing User :	Clive Harris
Date Deposited :	30 Oct 2019 12:38
Last Modified :	30 Oct 2019 12:38
URI:	http://epubs.surrey.ac.uk/id/eprint/852993

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