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.

The 356 nt noncoding satellite RNA C (satC) of Turnip crinkle virus (TCV) is composed of 5′ sequences from a second TCV satRNA (satD) and 3′ sequences derived from TCV. SHAPE structure mapping revealed that 76 nt in the poorly-characterized satD-derived region form an extended hairpin (H2). Pools of satC in which H2 was replaced with 76, 38, or 19 random nt were co-inoculated with TCV helper virus onto plants and satC fitness assessed using in vivo functional selection (SELEX). The most functional progeny satCs, including one as fit as wild-type, contained a 38-39 nt H2 region that adopted a hairpin structure and exhibited an increased ratio of dimeric to monomeric molecules. Some progeny of satC with H2 deleted featured a duplication of 38 nt, partially rebuilding the deletion. Therefore, H2 can be replaced by a 38-39 nt hairpin, sufficient for overall structural stability of the 5′ end of satC.

Turnip crinkle virus (TCV) contains a structured 3= region with hairpins and pseudoknots that form a complex network of noncanonical RNA:RNA interactions supporting higher-order structure critical for translation and replication. We investigated several second-site mutations in the p38 coat protein open reading frame (ORF) that arose in response to a mutation in the asymmetric loop of a critical 3= untranslated region (UTR) hairpin that disrupts local higher-order structure. All tested second-site mutations improved accumulation of TCV in conjunction with a partial reversion of the primary mutation (TCV-rev1) but had neutral or a negative effect on wild-type (wt) TCV or TCV with the primary mutation. SHAPE (selective 2=-hydroxyl acylation analyzed by primer extension) structure probing indicated that these second-site mutations reside in an RNA domain that includes most of p38 (domain 2), and evidence for RNA:RNA interactions between domain 2 and 3=UTR-containing domain 1 was found. However, second-site mutations were not compensatory in the absence of p38, which is also the TCV silencing suppressor, or in dcl-2/dcl4 or ago1/ago2 backgrounds. One second-site mutation reduced silencing suppressor activity of p38 by altering one of two GW motifs that are required for p38 binding to double-stranded RNAs (dsRNAs) and interaction with RNA-induced silencing complex (RISC)-associated AGO1/AGO2. Another second-site mutation substantially reduced accumulation of TCVrev1 in the absence of p38 or DCL2/DCL4. We suggest that the second-site mutations in the p38 ORF exert positive effects through a similar downstream mechanism, either by enhancing accumulation of beneficial DCL-produced viral small RNAs that positively regulate the accumulation of TCV-rev1 or by affecting the susceptibility of TCV-rev1 to RISC loaded with viral small RNAs. IMPORTANCEGenomes of positive-strand RNA viruses fold into high-order RNA structures. Viruses with mutations in regions critical for translation and replication often acquire second-site mutations that exert a positive compensatory effect through reestablishment of canonical base pairing with the altered region. In this study, two distal second-site mutations that individually arose in response to a primary mutation in a critical 3= UTR hairpin in the genomic RNA of turnip crinkle virus did not directly interact with the primary mutation. Although different second-site changes had different attributes, compensation was dependent on the production of the viral p38 silencing suppressor and on the presence of silencing-required DCL and AGO proteins. Our results provide an unexpected connection between a 3= UTR primary-site mutation proposed to disrupt higher-order structure and the RNA-silencing machinery.T he structure of the genomic RNA (gRNA) of positive-strand RNA viruses participates in many basic functions, including replication, translation, and regulation of the competing processes of translation and transcription (1-3). Studies examining the secondary structure and tertiary in...