Inter-segment complementarity in orbiviruses: a driver for co-ordinated genome packaging in the Reoviridae?

Abstract

The process by which eukaryotic viruses with segmented genomes select a complete set of genome segments for packaging into progeny virus particles is not understood. In this study a model based on the association of genome segments through specific RNA-RNA interactions driven by base pairing was formalized and tested in the Orbivirus genus of the Reoviridae family. A strategy combining screening of the genomic sequences for inter-segment complementarity with direct functional testing of inter-segment RNA-RNA interactions using reverse genetics is described in the type species of the Orbivirus genus, Bluetongue virus (BTV). Two examples, involving four of the ten BTV genomic segments, of specific intersegment interaction motifs whose maintenance is essential for the generation of infectious virus, were identified. Equivalent inter-segment complementarities were found between the identified regions of the orthologous genome segments of all orbiviruses, including phylogenetically distant species. Specific interaction of the participating RNA segments was confirmed in vitro using electrophoretic mobility shift assays, with the interactions inhibited using oligonucleotides complementary to the interaction motif of one of the interacting partners, and also through mutagenesis of the motifs. In each example, the base pairing rather than the absolute sequence was critical to the formation of a functional inter-segment interaction, with mutations only being tolerated in rescued virus if compensating changes were made in the interacting partner to restore uninterrupted base pairing. The absolute sequence of the complementarity motifs varied between species, indicating that this newly identified phenomenon may contribute to the observed lack of reassortment between Orbivirus species.

Co-evolution in a putative bundling signal of bluetongue and epizootic hemorrhagic disease viruses

“…The co-evolving pair of complementary nucleotide sequences between S5 and S10 identified in the present study corresponded to one of two putative bundling signals reported in the previous experimental study mentioned above (Boyce et al, 2016). This observation validates the effectiveness of the strategy of identifying coevolving pairs of complementary nucleotide sequences as candidates for bundling signals in viruses with segmented genomes (Suzuki, 2014(Suzuki, , 2015.…”

“…It has been reported that a pair of nucleotide sequences consisting of 6-11 bases located in the 5' end of the open reading frame (ORF) of S1 and in the 3' untranslated region (UTR) of S7, and also a pair of nucleotide sequences consisting of 6-8 bases located in the 5' UTR of S5 and in the 3' UTR of S10, were both complementary in each of 15 Orbivirus species, although the length and the absolute sequence of these complementary nucleotide sites varied among the species (Boyce et al, 2016). By predicting the intramolecular secondary structures for + RNAs of these segments in each species, the sites identified in S7 and S10 were inferred to be located in loops at the end of stable stems, whereas those identified in S1 and S5 were inferred to be located in regions of low base pairing (Boyce and McCrae, 2015;Boyce et al, 2016).…”

“…By predicting the intramolecular secondary structures for + RNAs of these segments in each species, the sites identified in S7 and S10 were inferred to be located in loops at the end of stable stems, whereas those identified in S1 and S5 were inferred to be located in regions of low base pairing (Boyce and McCrae, 2015;Boyce et al, 2016). In addition, the experi-mental studies using BTV demonstrated that the complementarities between S1 and S7 and between S5 and S10 were critical not only for interaction of + RNAs in vitro but also for production of infectious progeny virions in vivo (Boyce and McCrae, 2015;Boyce et al, 2016). These observations suggest that the two pairs of complementary nucleotide sequences are putative bundling signals of Orbivirus.…”

“…The genomes of BTV and EHDV consist of 10 double-stranded RNA (dsRNA) segments designated S1-S10, each of which encodes one or two proteins (Attoui et al, 2012;Roy, 2013). These viruses are considered to undergo selective packaging, in which one copy each of the 10 genomic segments is packaged in a virion (Lourenco and Roy, 2011;Sung and Roy, 2014;Boyce et al, 2016). The selective packaging is thought to be mediated by supramolecular complex formation of 10 positive-sense RNAs ( + RNAs) Fajardo et al, 2015), which are encapsidated in the core prior to the synthesis of negative-sense RNAs ( − RNAs) to generate dsRNAs (Lourenco and Roy, 2011;Sung and Roy, 2014).…”

“…Sites containing gaps were eliminated from the analysis. Only the Watson-Crick base pairs of U and A, and C and G were regarded as complementary, even though U and G may also form a wobble base pair, to reduce false positives in identifying functionally relevant pairs of complementary nucleotide sequences (Boyce et al, 2016). A pair of complementary single-nucleotide sites was considered to have co-evolved between BTV and EHDV when nucleotides at both sites in BTV isolates were all different from those in EHDV isolates.…”