Localizing the reovirus packaging signals using an engineered m1 and s2 ssRNA

Abstract

Using in vitro engineered and transcribed reovirus m1 and s2 ssRNAs, we demonstrate that the nucleotides used to identify these ssRNAs are localized to the 5' and not the 3' termini. To demonstrate this, we used our previously reported S2-CAT reovirus and we report the creation of an engineered M1-CAT reovirus. The M1 gene of this virus retains 124 nucleotides from the wild type M1 gene preceding the CAT gene and 172 nucleotides from the wild type gene following the CAT gene. The engineered M1-CAT ssRNA is 1048 nucleotides in length, much shorter than the wild type M1 at 2304 nucleotides. We have used a set of chimeric s2.m1 ssRNAs to localize the packaging signals within these RNAs. By packaging signals we mean that the presence of these signals in engineered ssRNAs results in these ssRNAs being replicated to dsRNA and packaged into progeny virus. An engineered ssRNA with a 5' sequence identical with the wild type s2 ssRNA, supported by a 3' sequence from either the m1 or s2 ssRNA, is incorporated into a virus as an S2 dsRNA. Likewise, an ssRNA with an m1 5' end is incorporated as an M1 dsRNA.