Structure-function analysis of the equine hepacivirus 5' untranslated region highlights the conservation of translational mechanisms across the hepaciviruses.

Joseph Lattimer,Andrew Tuplin,Hazel Stewart,Mark Harris,Nicolas Locker,Nicola J Stonehouse

doi:10.1099/jgv.0.001316

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.

Highlights

As obligate intracellular parasites, viruses rely on the host cell machinery for translation
This study provides the first experimental confirmation of the secondary structures within the Equine hepacivirus (EHcV) 5′untranslated regions (UTRs) (Fig. 2) and delineates the essential internal ribosome entry site (IRES) as consisting of SLIII and the adjacent pseudoknot, whilst the preceding SLI, SLIA and SLII are not required for minimal IRES activity
Whilst the deletion of SLI alone had no effect on translation efficiency, the absence of SLI and SLII together caused a significant impairment, indicating that SLII may contribute to IRES function indirectly through ribosomal contacts

Summary

Introduction

Viruses rely on the host cell machinery for translation. Viral IRES elements have been classified into six types, depending upon their structure and requirement for host cell factors, termed picornavirus type I–V IRESs and intergenic region IRESs [1,2,3,4,5,6,7]. Type IV IRESs are known as HCV-like IRESs, as the 5′ untranslated region (5′UTR) of hepatitis C virus (HCV), contains a series of RNA structures that cooperatively direct both ribosome assembly and initiation of cap-independent translation of the viral polyprotein. The 5′UTR of equine hepacivirus (EHcV, previously termed non-primate hepacivirus and classified as hepacivirus A), the most closely related virus to HCV, has been described to function as an IRES [8] and constitutes another type IV IRES. Investigating the replication mechanisms of this putative HCV model is important to identify which are the causative elements underlying these divergent pathologies

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Conclusion