Multiple Cis-acting elements modulate programmed -1 ribosomal frameshifting in Pea enation mosaic virus.

Feng Gao,Anne E Simon

doi:10.1093/nar/gkv1241

Abstract

Programmed -1 ribosomal frameshifting (-1 PRF) is used by many positive-strand RNA viruses for translation of required products. Despite extensive studies, it remains unresolved how cis-elements just downstream of the recoding site promote a precise level of frameshifting. The Umbravirus Pea enation mosaic virus RNA2 expresses its RNA polymerase by -1 PRF of the 5′-proximal ORF (p33). Three hairpins located in the vicinity of the recoding site are phylogenetically conserved among Umbraviruses. The central Recoding Stimulatory Element (RSE), located downstream of the p33 termination codon, is a large hairpin with two asymmetric internal loops. Mutational analyses revealed that sequences throughout the RSE and the RSE lower stem (LS) structure are important for frameshifting. SHAPE probing of mutants indicated the presence of higher order structure, and sequences in the LS may also adapt an alternative conformation. Long-distance pairing between the RSE and a 3′ terminal hairpin was less critical when the LS structure was stabilized. A basal level of frameshifting occurring in the absence of the RSE increases to 72% of wild-type when a hairpin upstream of the slippery site is also deleted. These results suggest that suppression of frameshifting may be needed in the absence of an active RSE conformation.

Highlights

The rapid, efficient life cycle of positive-strand RNA viruses requires optimal usage of their compact genomes for gene expression and RNA replication
Programmed ribosomal frameshifting (PRF) and translational readthrough are two ribosome recoding mechanisms employed by a large number of plant, fungal, animal- and human-infecting viruses to control when translation of an open reading frames (ORFs) terminates at a stop codon or carries on to produce a C-terminally extended polypeptide [5,6,7,8,9,10,11,12]
To investigate the role of nucleotides within and flanking the slippery sequence on -1PRF, mutations were introduced into full-length Pea enation mosaic virus (PEMV) gRNA (Figure 1B) and levels of p94 were assayed for in wheat germ extracts (WGE) (Figure 1C). gRNA containing mutations in the variable ‘GG’ just upstream of the p33 stop codon as well as in the flanking conserved ‘U’ had near WT levels of frameshifting (UGG-m1, UGG-m2 and UGG-m3)

Summary

Introduction

The rapid, efficient life cycle of positive-strand RNA viruses requires optimal usage of their compact genomes for gene expression and RNA replication. Programmed ribosomal frameshifting (PRF) and translational readthrough are two ribosome recoding mechanisms employed by a large number of plant-, fungal-, animal- and human-infecting viruses to control when translation of an ORF terminates at a stop codon or carries on to produce a C-terminally extended polypeptide [5,6,7,8,9,10,11,12]. Since the recoded product of many animal and plant viruses is the viral RNAdependent RNA polymerase (RdRp), maintaining a precise ratio of extension to termination products appears critical for efficient propagation of the virus within the host [6,8,15,16,17]. The recoding event must be strictly modulated during the viral life cycle

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Results

Conclusion