Cis-acting Element at the 5' Non-Coding Region of Tacaribe Virus S RNA Modulates Genome Replication

Abstract

Tacaribe virus (TCRV) is the prototype of New World mammarenaviruses, a group that 27 includes several members causing hemorrhagic fevers in humans. The TCRV genome 28 comprises two RNA segments named S (small) and L (large). Both genomic segments 29 contain non-coding regions (NCR) at their 5? and 3? ends. While the 5? and 3? terminal 19-nt 30 sequences are known to be essential for promoter function, the role of their neighboring 31 internal non-coding sequences (iNCR) remains poorly understood. To analyze the relevance 32 of the 5? and 3? iNCR in TCRV S RNA synthesis, mutant S-like minigenomes and 33 miniantigenomes were generated. Using a minireplicon assay, Northern blotting and RT-34 qPCR, we demonstrated that the genomic 5? iNCR is specifically engaged in minigenome 35 replication, yet is not directly involved in minigenome transcription, and showed that the S 36 genome 3? iNCR is barely engaged in this process. Analysis of partial deletions and point 37 mutations, as well as total or partial substitution of the 5? iNCR sequence led us to conclude 38 that the integrity of the whole genomic 5? iNCR is essential and that a local predicted 39 secondary structure or RNA-RNA interactions between the 5? and 3? iNCRs are not strictly 40 required for viral S RNA synthesis. Furthermore, we employed a TCRV reverse genetic 41 approach to ask whether manipulation of the S genomic 5? iNCR sequence may be suitable 42 for viral attenuation. We found that mutagenesis of the 5? promoter-proximal subregion 43 slightly impacts on recombinant TCRV virulence in vivo.