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Features Of The Mammalian Orthoreovirus 3 Dearing L1 Single-stranded RNA That Direct Packaging And Serotype Restriction

Michael R. Roner, Bradley G. Steele

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A series of recombinant mammalian orthoreoviruses (mammalian orthoreovirus 3 Dearing, MRV-3De) were generated that express an MRV-3De λ3–CAT fusion protein. Individual viruses contain L1CAT double-stranded (ds) RNAs that range in length from a minimum of 1020 bp to 4616 bp. The engineered dsRNAs were generated from in vitro-transcribed single-stranded (ss) RNAs and incorporated into infectious virus particles by using reverse genetics. In addition to defining the sequences required for these ssRNAs to be ‘identified’ as l1 ssRNAs, the individual nucleotides in these regions that ‘mark’ each ssRNA as originating from mammalian orthoreovirus 1 Lang (MRV-1La), mammalian orthoreovirus 2 D5/Jones (MRV-2Jo) or MRV-3De have been identified. A C at position 81 in the MRV-1La 5′ 129 nt sequence was able to be replaced with a U, as normally present in MRV-3De; this toggled the activity of the MRV-1La ssRNA to that of an MRV-3De 5′ l1. RNA secondary-structure predictions for the 5′ 129 nt of both the biologically active MRV-3De l1 ssRNA and the U81-MRV-3De-restored MRV-1La 5′ ssRNA predicted a common structure.