Conserved non-coding RNA motifs influence the neuropathogenicity of Simbuviruses: Molecular dissection in the Schmallenberg virus model

The Simbu serogroup, part of the Peribunyaviridae family, includes arboviruses associated with febrile illnesses in humans and fetal congenital malformations due to viral neurotropism in ruminants. These viruses possess a tripartite, negative-sense RNA genome lacking the poly(A) tail. Notably, the 5' untranslated region (UTR) of the small (S) genomic segment contains conserved RNA elements, including a stem-loop (SL) structure and a sequence-based motif (GC signal) flanking the messenger RNA (mRNA) termination site. Although their functions remain unclear, their conservation and specific location suggest a potential role in mRNA transcription termination and translation initiation. A reverse genetics system for Schmallenberg virus (SBV) was used to create a viral recombinant library bearing deliberate mutations in both motifs. Replication kinetics, S segment transcription termination, and Nucleocapsid protein (N) abundance of rescued virus mutants were evaluated in mammalian and insect cell culture. Virulence was assessed in an immunocompetent mouse model. Characterization of the mutant viruses indicated that the SL structure is essential for viral production, with the stem length as a key feature; more than three complementary base pairs between the stem arms are necessary for replication. A shorter stem length impaired replicative fitness, N protein abundance and altered the mRNA to genomic RNA ratio. Point mutations in the GC signal disrupted proper mRNA termination, thereby limiting viral N protein synthesis and, thus, virion assembly. In vivo, attenuated viruses resulted in lower viral loads, reduced dissemination in mice brains, and improved survival rates compared to wild-type SBV. The GC signal mutants exhibited strong attenuation while still maintaining active transcription. Overall, these findings indicate that the SL and GC signal serve as cis-regulatory elements and are indirect determinants of SBV virulence, regulating viral replication and influencing neuropathogenesis.