Clarifying the role of RNA interactions in RVA genetic stability and evolution

Group A rotaviruses (RVAs) account for approximately 200,000 child fatalities each year. Their genomes consist of 11 unique doublestranded RNA segments that evolve through reassortment. Engineered RVA strains frequently exhibit genomic rearrangements, hindering the development of potential vaccine candidates. We hypothesise that RNA-RNA

incompatibilities resulting from stable base-pairing contribute to this instability. We propose to understand and address the mechanisms. Aims: 1.

Identify the sources of genomic instability in recombinant RVA strains including heterologous DNA segments. 2. Apply RNA-structure informed sequence optimization to enhance the stability of recombinant RVAs. Aim 1: We will determine whether genome rearrangements arise during replication or transcription.

Transcription assays will be performed using double-layered particles (DLPs) derived from recombinant RVAs with heterologous inserts that induce genetic instability. Direct nanopore sequencing of individual transcripts (with the Keyser group) will reveal the prevalence of aberrant transcripts, transcription rates of heterologous sequences, and the frequency of incomplete transcription events.

These analyses will indicate the molecular foundations genetic instability of RVA, and give insights into viral transcription regulation. Aim 2: Using RNA cross-linking (SPLASH), we will discover how heterologous genetic inserts influence inter- and intra-molecular RNA-RNA contacts in RVA genomes. Mutations introduced at interaction sites will be tested for effects on replication kinetics and genetic stability and the conclusions used to refine the strategy.

Outcome: These studies will clarify the role of RNA interactions in RVA genetic stability and evolution. Insights and tools developed in this project may offer strategies to stabilise other RNA viruses, including influenza.