The 3′ Untranslated Region of Pea Enation Mosaic Virus Contains Two T-Shaped, Ribosome-Binding, Cap-Independent Translation Enhancers

Abstract

Many plant viruses without 5=caps or 3= poly(A) tails contain 3= proximal, cap-independent translation enhancers (3=CITEs) that bind to ribosomal subunits or translation factors thought to assist in ribosome recruitment. Most 3=CITEs participate in a longdistance kissing-loop interaction with a 5= proximal hairpin to deliver ribosomal subunits to the 5= end for translation initiation. Pea enation mosaic virus (PEMV) contains two adjacent 3=CITEs in the center of its 703-nucleotide 3= untranslated region (3=UTR), the ribosome-binding, kissing-loop T-shaped structure (kl-TSS) and eukaryotic translation initiation factor 4E-binding Panicum mosaic virus-like translation enhance (PTE). We now report that PEMV contains a third, independent 3=CITE located near the 3= terminus. This 3=CITE is composed of three hairpins and two pseudoknots, similar to the TSS 3=CITE of the carmovirus Turnip crinkle virus (TCV). As with the TCV TSS, the PEMV 3=TSS is predicted to fold into a T-shaped structure that binds to 80S ribosomes and 60S ribosomal subunits. A small hairpin (kl-H) upstream of the 3=TSS contains an apical loop capable of forming a kissing-loop interaction with a 5= proximal hairpin and is critical for the accumulation of full-length PEMV in protoplasts. Although the kl-H and 3=TSS are dispensable for the translation of a reporter construct containing the complete PEMV 3=UTR in vitro, deleting the normally required kl-TSS and PTE 3=CITEs and placing the kl-H and 3=TSS proximal to the reporter termination codon restores translation to near wild-type levels. This suggests that PEMV requires three 3=CITEs for proper translation and that additional translation enhancers may have been missed if reporter constructs were used in 3=CITE identification. IMPORTANCEThe rapid life cycle of viruses requires efficient translation of viral-encoded proteins. Many plant RNA viruses contain 3= capindependent translation enhancers (3=CITEs) to effectively compete with ongoing host translation. Since only single 3=CITEs have been identified for the vast majority of individual viruses, it is widely accepted that this is sufficient for a virus's translational needs. Pea enation mosaic virus possesses a ribosome-binding 3=CITE that can connect to the 5= end through an RNA-RNA interaction and an adjacent eukaryotic translation initiation factor 4E-binding 3=CITE. We report the identification of a third 3=CITE that binds weakly to ribosomes and requires an upstream hairpin to form a bridge between the 3= and 5= ends. Although both ribosome-binding 3=CITEs are critical for virus accumulation in vivo, only the CITE closest to the termination codon of a reporter open reading frame is active, suggesting that artificial constructs used for 3=CITE identification may underestimate the number of CITEs that participate in translation.

Differential use of 3’CITEs by the subgenomic RNA of Pea enation mosaic virus 2

“…In addition, only the kl-TSS and PTE were required to significantly enhance translation of luciferase reporter constructs containing the PEMV2 full-length 3′UTR and the 5′ end 89-nt, which includes hairpin 5H2, the hairpin that participates in the long-distance interaction with the kl-TSS (Gao et al , 2012; Gao et al , 2014). However, the 3′TSS was important for accumulation of PEMV2 gRNA in protoplasts (Gao et al, 2014). In addition, when reporter constructs’ 3′UTR contained a deletion that removed the kl-TSS and PTE and placed the 3′TSS proximal to the luciferase termination codon, the 3′TSS enhanced translation in conjunction with a nearby hairpin that base-pairs with 5′ hairpin 5H2.…”

“…Among them, only the kl-TSS and the adjacent PTE contribute to translation of the full-length gRNA in WGE (Du et al , 2017), as well as translation of reporter constructs with 5′ gRNA sequences and the full-length 3′UTR in protoplasts (Gao et al , 2014). The 3′TSS, while not important for gRNA translation in these assays, is important for accumulation of the gRNA in protoplasts (Gao et al , 2014). In addition, the 3′TSS can enhance translation of luciferase reporter constructs missing the kl-TSS and PTE, with the long-distance interaction provided by a 3′TSS-proximal hairpin (Gao et al , 2014).…”

“…3′CITEs mainly facilitate translation by recruiting translation initiation factor eIF4F, via binding to its eIF4E and/or eIF4G subunits, followed by attraction of 40S subunits (Das Sharma et al , 2015; Gazo et al , 2004; Nicholson et al , 2010; Nicholson et al , 2013; Treder et al , 2008; Wang et al , 2009). In addition, some 3′CITEs can directly bind ribosomes or ribosomal subunits (Das Sharma et al , 2015; Gao et al , 2012; Gao et al , 2014; Stupina et al , 2008). Recruited translation elements are usually delivered to the 5′ end of the gRNA via a long-distance RNA:RNA interaction between the terminal loop of a hairpin associated with the 3′CITE and accessible sequences in the 5′UTR or nearby coding region (Chattopadhyay et al , 2011; Fabian and White, 2004, 2006; Gao et al , 2012; Nicholson and White, 2008; Nicholson et al , 2010; Simon and Miller, 2013; Wu et al , 2009).…”

“…Another virus with multiple 3′CITEs is the umbravirus Pea enation mosaic virus 2 (PEMV2), which has three 3′CITEs in its unusually long (703 nt) 3′UTR (Gao et al , 2014). These 3′CITEs are: (1) the kissing-loop T-shaped structure (kl-TSS), which binds 80S ribosomes and 40S and 60S ribosomal subunits and engages in a long-distance RNA:RNA interaction with a 5′ end hairpin (5H2) in the coding region of the 5′ proximal ORF (Gao et al , 2012); (2) the PTE, which is located just downstream of the kl-TSS and binds to eIF4E (Wang et al , 2009); and (3) the 3′TSS, which is also capable of binding to 80S ribosomes and 60S ribosomal subunits and is similar to the well-studied TSS of carmovirus Turnip crinkle virus (Gao et al , 2014; Stupina et al , 2008) (Fig. 1B).…”

“…1B). Only the kl-TSS and PTE were required for translation of full-length PEMV2 gRNA in wheat germ extracts (WGE) (Du et al , 2017), or reporter constructs containing the entire 3′UTR of PEMV2 (Gao et al , 2014) and thus the functional role of the 3′TSS is unknown.…”