Structural constraints in the packaging of bluetongue virus genomic segments

McCrae

Journal of General Virology

et al. 2016

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.

Section: Identification Of Potentially Functional Inter-segment Complmentioning

confidence: 99%

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

McCrae

Journal of General Virology

et al. 2016

“…Considering the effectiveness of inferring that co-evolving pairs of complementary nucleotide sequences are involved in biologically relevant interactions, as observed above, it is feasible that the stem has been functionally constrained during the evolution of BTV and EHDV. These results indicate that the intermolecular base pairing by the putative bundling signal between S5 and S10 may be facilitated by stem-loop structures within + RNAs of both genomic segments (Burkhardt et al, 2014;Sung and Roy, 2014;Boyce and McCrae, 2015). It is plausible that the loop exposes the complementary nucleotide sequence to make it available for intermolecular interaction, whereas the stem delineates the loop to ensure the specificity of intermolecular interaction.…”

mentioning

confidence: 89%

“…The supramolecular complex of + RNAs is thought to be formed by intermolecular base pairing of complementary nucleotide sequences termed the bundling signal (Goto et al, 2013). Intramolecular secondary structures within + RNAs, particularly stem-loop structures, are thought to facilitate intermolecular base pairing (Burkhardt et al, 2014;Sung and Roy, 2014;Boyce and McCrae, 2015).…”

mentioning

confidence: 99%

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

Suzuki

2016

Genes Genet. Syst.

Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) possess a genome of 10 segmented RNAs (S1-S10), one copy of each of which is considered to be packaged in a virion. This selective packaging is thought to be mediated by supramolecular complex formation of the 10 RNAs, through intermolecular base pairing of complementary nucleotide sequences termed the bundling signal. Here, the whole genomic sequences of BTV and EHDV isolates were analyzed to identify co-evolving pairs of complementary nucleotide sequences within and between genomic segments. One co-evolving pair was identified within S5 and another between S5 and S10. The co-evolving pair between S5 and S10, consisting of six bases in each segment, was a candidate for a bundling signal and was identical to one of two putative bundling signals reported in a previous experimental study, validating the effectiveness of the method used in the present study. The six bases in S10 were confirmed to be located in a loop at the end of a stable stem. Although the six bases in S5 were located in a loop at the end of a stem of only four bases long, the complementary nucleotide sequences constituting this stem were, remarkably, the co-evolving pair within S5. These results highlight the importance not only of loops but also of stems in the intermolecular base pairing of bundling signals.

Vector-Borne and Zoonotic Diseases

“…Still, the function of individual orbivirus proteins during replication requires further research. It is still not fully understood what proteins and which genomic regions are involved in orbivirus assembly and packaging (Burkhardt et al 2014). Recently, it was shown that the NS3/NS3a protein is not essential (van Gennip et al 2014), whereas RNA sequences in the NS3 gene are essential for BTV replication (Feenstra et al 2014b).…”

Section: Viral Genome Sequence and Encoded Proteinsmentioning

confidence: 99%

A Review of Knowledge Gaps and Tools for Orbivirus Research

Drolet

Rijn

Howerth

et al. 2015

Although recognized as causing emerging and re-emerging disease outbreaks worldwide since the late 1800 s, there has been growing interest in the United States and Europe in recent years in orbiviruses, their insect vectors, and the diseases they cause in domestic livestock and wildlife. This is due, in part, to the emergence of bluetongue (BT) in northern Europe in 2006-2007 resulting in a devastating outbreak, as well as severe BT outbreaks in sheep and epizootic hemorrhagic disease (EHD) outbreaks in deer and cattle in the United States. Of notable concern is the isolation of as many as 10 new BT virus (BTV) serotypes in the United States since 1999 and their associated unknowns, such as route of introduction, virulence to mammals, and indigenous competent vectors. This review, based on a gap analysis workshop composed of international experts on orbiviruses conducted in 2013, gives a global perspective of current basic virological understanding of orbiviruses, with particular attention to BTV and the closely related epizootic hemorrhagic disease virus (EHDV), and identifies a multitude of basic virology research gaps, critical for predicting and preventing outbreaks.