PARTICIPATION OF INTERNAL SEQUENCES WITHIN THE 5? AND 3? NON CODING REGIONS IN ARENAVIRUS RNA TRANSCRIPTION AND REPLICATION

D'ANTUONO, ALEJANDRA; FOSCALDI SABRINA; LOPEZ NORA

Siena

Congreso; XVI Negative Strand Viruses Meeting; 2015

NSV Meeting Organizing Committee

Abstract: Tacaribe virus (TCRV) is the prototype of the New World group of the Arenaviridae family. The TCRV genome comprises two segments named S (small) and L (large), each encoding two proteins in an ambisense orientation separated by an intergenic region. The RNA segments contain untranslated regions (UTRs) at their 5? and 3? ends whose length ranges from 30 to 77 nucleotides (nt). The terminal 19-nt sequence at the 3? end, which is highly conserved in each segment and across arenaviruses, shows sequence complementarity to the corresponding 5? terminal sequence allowing the formation of a panhandle structure. While these terminal sequences are known to contain signals required to promote viral polymerase binding and activity, the role of internal regions within the 3? and 5? UTR remains poorly understood. Using a TCRV S-like minigenome assay, we found that internal deletions within the genomic 5? UTR were associated with a strong inhibition in the synthesis of minigenome and anti-minigenome RNAs without affecting the relative proportion of mRNA. In contrast, internal deletions within the genomic 3? UTR had a less pronounced effect. Substitution of the entire internal sequence at the 5? UTR by the corresponding region from other arenaviruses did not affect the RNA minigenome/mRNA levels. In addition, when a conserved stem-loop structure predicted at the genomic 5′ UTR was deleted, the levels of minigenome expression were dramatically reduced. Similar results were observed upon deletion of an 8-nt sequence located nearby the predicted stem-loop. Substitution of this short sequence with an unrelated sequence partially restored minigenome levels, while mutagenesis of nucleotides predicted to stabilize the stem-loop structure had no effect. Overall, our results suggest that the internal sequence within the genomic 5? UTR is involved in viral RNA replication and as a consequence in mRNA transcription.