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

D´ANTUONO, ALEJANDRA; GALLO, GIOVANNA; SEPÚLVEDA, CLAUDIA; FERNÁNDEZ, JONÁS; BRIGNONE, JULIA; GAMBOA, GRACIELA; RIERA, LAURA; SAAVEDRA, M DEL CARMEN; LÓPEZ, NORA

JOURNAL OF VIROLOGY

AMER SOC MICROBIOLOGY

Lugar: Washington; Año: 2023

0022-538X

Tacaribe virus (TCRV) is the prototype of New World mammarenaviruses, a group that includes several members causing hemorrhagic fevers in humans. The TCRV genome comprises two RNA segments named S (small) and L (large). Both genomic segments contain non-coding regions (NCR) at their 5’ and 3’ ends. While the 5’ and 3’ terminal 19-nt sequences are known to be essential for promoter function, the role of their neighboring internal non-coding sequences (iNCR) remains poorly understood. To analyze the relevance of the 5’ and 3’ iNCR in TCRV S RNA synthesis, mutant S-like minigenomes and miniantigenomes were generated. Using a minireplicon assay, Northern blotting and RT35 qPCR, we demonstrated that the genomic 5’ iNCR is specifically engaged in minigenome replication, yet is not directly involved in minigenome transcription, and showed that the S genome 3’ iNCR is barely engaged in this process. Analysis of partial deletions and point mutations, as well as total or partial substitution of the 5’ iNCR sequence led us to conclude that the integrity of the whole genomic 5’ iNCR is essential and that a local predicted secondary structure or RNA-RNA interactions between the 5’ and 3’ iNCRs are not strictly required for viral S RNA synthesis. Furthermore, we employed a TCRV reverse genetic approach to ask whether manipulation of the S genomic 5’ iNCR sequence may be suitable for viral attenuation. We found that mutagenesis of the 5’ promoter-proximal subregion slightly impacts on recombinant TCRV virulence in vivo.