A novel factor-dependent internal ribosome entry site and programmed frameshifting signal in the Bemisia-associated dicistrovirus 2

Internal ribosome entry sites (IRES) are structured RNA elements that recruit ribosomes using a subset of translation factors in a 5' cap-independent manner. The dicistrovirus IRES uses the most streamlined mechanism: the IRES recruits ribosomes directly without the use of initiation factors and directs translation from a non-AUG codon. The IRES adopts three pseudoknots, stem-loop, and unpaired regions that interact with specific domains of the ribosomal 40S and 60S subunits. Analysis of dicistrovirus-like genomes identified via metagenomic studies predicts potentially novel atypical IRES RNA structures. Specifically, the Bemisia-associated dicistrovirus 2 (BaDV-2) IRES contains an unusual RNA structure that lacks conserved elements and altered structures that do not conform to the typical dicistrovirus IRES mechanism. Moreover, the BaDV-2 genome contains a predicted -1 frameshifting signal (FSS) with an RNA stem-loop structure that would direct the translation of the RNA-dependent RNA polymerase (RdRp) motif. In this study, using a bicistronic reporter, we demonstrate that BaDV-2 IGR supports internal ribosome entry activity in in vitro insect Sf-21 and rabbit reticulocyte translation extracts. Using deletion and mutagenesis analyses, we showed that the BaDV-2 IRES activity is within a minimal 140 nucleotide element containing a predicted stem loop. Moreover, the IRES is sensitive to eIF2 and eIF4A inhibitors, NSC1198983 and hippuristanol, respectively, indicating that the BaDV-2 IRES is factor-dependent, unlike typical factor-less dicistrovirus IRESs. Preliminary data shows that a chimeric dicistrovirus infectious clone does not support virus infection in Drosophila cells. Finally, we show that the predicted stem-loop structure -1 frameshifting signal can direct programmed frameshifting in vitro using a luciferase-based bicistronic reporter. In summary, we have provided evidence of the first -1 frameshifting signal and a novel IRES mechanism in this viral family, thus highlighting the diversity of viral strategies to direct viral protein synthesis.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat