title: Generation of infectious RNA complexes in Orbiviruses: RNA-RNA interactions of genomic segments.
abstract: Viruses with segmented RNA genomes must package the correct number of segments for synthesis of infectious virus particles. Recent studies suggest that the members of the Reoviridae family with segmented double-stranded RNA genomes achieve this challenging task by forming RNA networks of segments prior to their recruitment into the assembling capsid albeit direct evidence is still lacking. Here, we investigated the capability of virus recovery by preformed complexes of ten RNA segments of Epizootic Haemorrhagic Disease Virus (EHDV), a Reoviridae member, by transcribing exact T7 cDNA copies of genomic RNA segments in a single in vitro reaction followed by transfection of mammalian cells. The data obtained was further confirmed by RNA complexes generated from Bluetongue virus, another family member. Formation of RNA complexes was demonstrated by sucrose gradient ultracentrifugation, and RNA-RNA interactions inherent to the formation of the RNA complexes were demonstrated by electrophoretic mobility shift assay. Further, we showed that disruption of RNA complex formation inhibits virus recovery, confirming that recruitment of complete RNA networks is essential for packaging and consequently, virus recovery. This efficient reverse genetics system will allow further understanding of evolutionary relationships of Reoviridae members and may also contribute to development of antiviral molecules.
authors.affiliation: Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, United Kingdom.
authors.forename: Teodoro
authors.initials: T
authors.lastname: Fajardo
authors.affiliation: Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, United Kingdom.|Current address: Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Kinda
authors.initials: K
authors.lastname: AlShaikhahmed
authors.affiliation: Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, United Kingdom.
authors.forename: Polly
authors.initials: P
authors.lastname: Roy
mesh_terms: D000818:Animals; D016679:Genome, Viral / Q000235:genetics*; D023281:Genomics / Q000379:methods*; D006801:Humans; D018163:Orbivirus; D012330:RNA, Double-Stranded / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D014779:Virus Replication
chemical_list: D012330:RNA, Double-Stranded; D012367:RNA, Viral
keywords: Immune response; Immunity; Immunology and Microbiology Section; RNA-RNA interactions; dsRNA virus; orbivirus genome assembly; reverse genetics; segmented genome sorting and packaging