Arenavirus mRNAs defy the central dogma of eukaryotic gene expression by achieving stability and efficient translation without the poly(A) tail and with short 5’UTRs, long considered essential for both processes. Despite the growing public health threat posed by arenaviruses like Lassa virus (LASV), the molecular basis for its unconventional stability and translation strategies remains poorly understood. To elucidate the molecular basis of these mechanisms, we applied Nanopore direct RNA sequencing to first define the precise termini of LASV transcripts. These analyses revealed the presence of compact, GC-rich intergenic regions (IGRs) at the 3’ end of LASV mRNAs forming conserved stem-loop structures and short, structured 5′ untranslated regions (UTRs). In addition, we uncovered a strong preference for U-rich cap-snatched sequences in NP and GPC mRNAs. Using luciferase reporters that incorporate these sequence and structural features, we found that the LASV NP 3′ IGR enhances mRNA stability up to six-fold compared to polyadenylated controls, while the native 5′ UTR drives strong, cap-dependent translation even in the absence of a poly(A) tail. Synthetic IGR variants confirmed that GC content and predicted folding energy correlate with increased resistance from 3′→5′ exonuclease decay. Finally, degron-based screens identified essential host translation factors that drive the non-canonical translation of arenavirus mRNAs.

Together, these findings define how LASV mRNAs encode their own stability and translation logic through structured RNA elements independent of canonical poly(A)-dependent interactions. This mechanism expands our understanding of non-canonical gene expression and suggests new design principles for broad antivirals that target the unique features of arenavirus mRNAs.