Po-yu Sung, London School of Hygiene and Tropical Medicine

Po-Yu Sung(1), Jody E Phelan(1), Dongsheng Luo(1), Redmond P Smyth(2), Polly Roy(1) 1. Department of Infection Biology, London School of Hygiene and Tropical Medicine, London, UK. 2. Institute of Molecular and Cellular Biology, Strasbourg, France

Virus Replication: Entry, Exit and Everything in Between

How do segmented double-stranded (ds) RNA viruses correctly recruit their genome has yet to be clarified. Both bluetongue virus (BTV) and rotavirus have genomes of dsRNA segments of different sizes, but they assemble their genome in single-stranded (ss) form through a series of specific RNA-RNA interactions prior to packaging. We used 2'-hydroxyl acylation analysed by primer extension and mutational profiling (SHAPE-MaP) to identify RNA structural changes during complex formation and putative RNA-RNA interaction sites. Our data revealed a core RNA-complex of smaller segments which serves as the foundation for the assembly of complete network composed of ten BTV ssRNA segments. No viral protein was required for these assembly reactions. To investigate whether this scenario also occurs in infected live cells, we used nanopore dimethylsulfate mutational profiling (Nano-DMS-MaP) to analyse BTV and rotavirus infected cells, during their early stage of replication. Formation of ssRNAs complex was observed in both BTV and rotavirus infection, indicating a similarity in the genome assembly mechanism. However, we found differences in segments interacting pattern in two viruses. With reverse genetics method for both BTV and rotavirus, we demonstrated that substitution silent mutations in the interacting bases affected virus replication. These data prove the principle of a ssRNA driven reaction that may apply to various of dsRNA viruses, which may offer novel opportunities for designed attenuation or antiviral therapeutics.