Saguaro cactus virus (SCV) and Turnip crinkle virus (TCV) are two plant positive-strand [(+)-strand] RNA carmoviruses that serve as model systems for studying long- and short-distance RNA:RNA interactions required for essential viral processes in host cells. Our search for conserved 3’ cap-independent translational elements (3’ CITE) in the carmovirus genus led to the discovery of a Panicum mosaic virus-like 3’CITE (PTE) in seven carmoviruses, including SCV. We found that the SCV PTE participates in long-distance kissing-loop interactions with hairpins located in the p26 open reading frame (ORF) of the genomic RNA (gRNA) and in the 5’ untranslated region (UTR) of the small subgenomic RNA (sgRNA2), and promotes efficient translation of both gRNA and sgRNA2 reporter mRNAs in plant protoplasts. For nearly all PTE-containing carmoviruses, a 5’ hairpin that participates in a kissing-loop interaction with the PTE is found in conserved locations in the 5’ ends of the gRNA and sgRNA2. We found that moving the 5’ hairpin 27 nt closer to the 5’ end had only a minimal effect on both gRNA and sgRNA2 translation. However, moving the 5’ hairpin further in the direction of the 5’ end had a detrimental effect.

The higher order structure of the TCV 3’ region is supported by a complex network of canonical and noncanonical medium- and short-distance RNA:RNA interactions that modulate many critical viral processes. We investigated several second-site mutations in the coat protein (p38) ORF that arose in response to a primary-site mutation (m21) located in critical 3’UTR hairpin H4. All tested second-site mutations were compensatory with a partial reversion of m21 (TCV-rev1). Selective 2’-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) structure probing indicated that these second-site mutations in the p38 ORF reside in a separate RNA domain (Domain2) and provided evidence for RNA:RNA interactions between Domain2 and 3’UTR-containing Domain1. These second-site mutations, however, were not compensatory in the absence of p38, the TCV silencing suppressor, or in the absence of functional Dicer-like proteins (DCLs) and RNA induced silencing complex (RISC). Our results indicate an unexpected connection between a 3’ primary-site mutation and the RNA silencing machinery in TCV.