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

## TL;DR

This study shows that specific RNA structures in Schmallenberg virus are crucial for its replication and ability to cause neurological disease in mice.

## Contribution

The study identifies conserved RNA motifs as key regulators of SBV replication and neuropathogenicity using reverse genetics and in vivo models.

## Key findings

- The stem-loop structure is essential for viral replication, with stem length directly affecting replicative fitness and N protein abundance.
- Mutations in the GC signal disrupt mRNA termination and N protein synthesis, impairing virion assembly.
- GC signal mutants showed strong attenuation in mice, with reduced brain dissemination and improved survival.

## Abstract

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.

Understanding how viruses control their gene expression and cause disease is crucial for improving control strategies. Our research focuses on Schmallenberg virus, a ruminant pathogen. The virus belongs to a group of viruses that can cause congenital disorders when pregnant females transmit the virus to the developing fetal nervous system. We were interested in specific viral genome conserved features suspected to be crucial for the virus’s life cycle. These include a structural element (stem-loop) and a sequence-based element, both found in similar viruses. Adopting a reverse genetics system we created modified versions of SBV to see how these elements impacted the virus infection in cells and mouse brains. Our findings showed that the mutant viruses produced fewer viral proteins and replicated poorly in cells. In mice, these altered viruses demonstrated reduced replication and spread in the brain, leading to mild disease and less mortality. Our results confirmed that the non-coding RNA structural element is essential for the virus to complete its life cycle, specifically the stem length being significant. The sequence-based element is also vital for properly processing viral genetic material and protein synthesis in the infected cells. These findings provide new insights into how these viruses operate and may identify potential targets for developing vaccines or improved strategies for controlling them.