PhD thesis defense in veterinary sciences by Laura RUBIANO BONIL

Are non-coding Simbuviruses RNA motifs involved in transcription termination and translation?

Candidate

Laura RUBIANO BONIL

Promoters

Dr. Damien COUPEAU, UNamur, Department of veterinary medicine, Integrated Veterinary Research Unit (URVI) - promoter

Prof. Benoît MUYLKENS, UNamur, Department of veterinary medicine, Integrated Veterinary Research Unit (URVI) - co-promoter

Jury

• Dr. Damien Coupeau, University of Namur, Belgium (promoter)
• Prof. Benoît Muylkens, University of Namur, Belgium (co-promoter)
• Dr. Patricia Renard, University of Namur, Belgium
• Dr. Damien Vitour, Agency for Food, Environmental and Occupational Health & Safety (ANSES), France
• Dr. Charles Nicaise, University of Namur, Belgium
• Dr. Ludovic Martinelle, University of Liège, Belgium
• Dr. Lionel Tafforeau, University of Mons, Belgium

Summary

The Simbu serogroup, part of the Peribunyaviridae family, includes arboviruses associated with febrile disease and fetal congenital malformations due to viral neurotropism. These viruses possess a tripartite, negative-sense RNA genome that lacks the poly(A) tail. Notably, the 3' non-translated region 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 termination site for messenger RNA (mRNA) synthesis. Although their functions remain unclear, their conservation and specific positions suggest a potential role in mRNA transcription termination and translation initiation. A reverse genetics system for Schmallenberg virus (SBV), a ruminant pathogen, was used to generate a viral recombinant library bearing deliberate mutations. The replication kinetics, S segment transcription termination profile, and nucleoprotein (N) abundance were evaluated in mammalian and insect cell lines. At the same time, the 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 being a key feature; at least five complementary base pairs are necessary between the stem arms. A shorter stem length impaired replicative fitness, N protein abundance, and altered the mRNA to complementary 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 neuroinvasion, and improved survival rates compared to the 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 neuropathogenesis.