Plus-strand RNA viruses serve as templates for translation and then transcription by newly synthesized RdRp. A ribosome-binding tRNA-shaped structure (TSS) and upstream hairpin H4 in the 3′ UTR of Turnip crinkle virus (TCV) play key roles in translation and transcription. Second-site mutations generated to compensate for altering the critical asymmetric internal loop of H4 included a three-to-two base alteration in the terminal loop of a 3′ proximal hairpin (Pr) located downstream of the TSS. Unlike the non-deleterious three base alteration, single mutations in Pr loop were detrimental for RdRp transcription while enhancing translation and RdRp binding. One deleterious mutation in the Pr loop altered the structures of both the TSS and H4. These complex interactions in the 3′ UTR support a compact structural arrangement likely permitting RdRp access to a number of residues within a 195-base region including the 3′ end that are necessary for efficient transcription initiation.

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 contains a T-shaped, ribosome-binding, translation enhancer (TSS) in its 3’UTR that serves as a hub for interactions throughout the region. The viral RNA-dependent RNA polymerase (RdRp) causes the TSS/surrounding region to undergo a conformational shift postulated to inhibit translation. Using optical tweezers (OT) and steered molecular dynamic simulations (SMD), we found that the unusual stability of pseudoknotted element H4a/Ψ3 required five upstream adenylates, and H4a/Ψ3 was necessary for cooperative association of two other hairpins (H5/H4b) in Mg2+. SMD recapitulated the TSS unfolding order in the absence of Mg2+, showed dependence of the resistance to pulling on the 3D orientation and gave structural insights into the measured contour lengths of the TSS structure elements. Adenylate mutations eliminated one-site RdRp binding to the 3’UTR, suggesting that RdRp binding to the adenylates disrupts H4a/Ψ3, leading to loss of H5/H4b interaction and promoting a conformational switch interrupting translation and promoting replication.DOI: http://dx.doi.org/10.7554/eLife.22883.001

Next-Generation Sequencing is a rapidly advancing technology that has research and clinical applications. For many cancers, it is important to know the precise mutation(s) present, as specific mutations could indicate or contra-indicate certain treatments as well as be indicative of prognosis. Using the Ion Torrent Personal Genome Machine and the AmpliSeq Cancer Hotspot panel v2, we sequenced two pancreatic cancer cell lines, BxPC-3 and HPAF-II, alone or in mixtures, to determine the error rate, sensitivity, and reproducibility of this system. The system resulted in coverage averaging 2000× across the various amplicons and was able to reliably and reproducibly identify mutations present at a rate of 5%. Identification of mutations present at a lower rate was possible by altering the parameters by which calls were made, but with an increase in erroneous, low-level calls. The panel was able to identify known mutations in these cell lines that are present in the COSMIC database. In addition, other, novel mutations were also identified that may prove clinically useful. The system was assessed for systematic errors such as homopolymer effects, end of amplicon effects and patterns in NO CALL sequence. Overall, the system is adequate at identifying the known, targeted mutations in the panel.